IJDIES  ON  BACTERIUM  COLI 
1  CLOSELY  RELATED  FORMS 


UC-NRLF 


BY 


MAX/LEVINE 


THESIS 

Submitted  in  Partial  Fuliillmc-iil   of  the  Requirements 

for  the  I). 


DOCTOR  OF  PHILOSOPHY 
IN  BACTERIOLOGY 


In 


THE  GRADrATE  SCHOOL 

of  the 

UNIVERSITY    OF  IOWA 
1922 


EXCHANGE 


BiOLOGY 

Jf-i-.- 
6 


STUDIES  ON  BACTERIUM  CCLI 
CLOSELY  RELATED  FORMS 


BY 


MAX  LEVINE 


THESIS 

Submitted  in  Partial  Fulfillment  of  the  Requirements 
for  the  Degree  of 


DOCTOR  OF  PHILOSOPHY 
IN  BACTERIOLOGY 


In 


THE  GEADUATE  SCHOOL 

of  the 

UNIVEESITY  OF  IOWA 
1922 


„.,    .- 

G 


CONTENTS 


Notes  on  Bact.  coli  and  Bact.  aerogenes.  Journal  of  the  American 
Public  Health  Association,  1921,  Vol.  11,  pp.  31-34. 

Acid  Production  and  Other  Characteristics  of  B.  coli-like  bacteria 
from  Feces  and  Sewage.  Journal  of  Infectious  Diseases,  1917, 
Vol.  19,  pp.  773-805. 

A  Statistical  Classification  of  the  Colon  Cloacae  Group.  Journal 
of  Bacteriology,  1918,  Vol.  3,  pp.  253-276. 

Differentiation  of  Bact.  coli  and  Bact.  aerogenes  on  Solid  (a  Sim- 
plified Eosine  Methylene-blue  Agar)  Media.  Journal  of  In- 
fectious Diseases,  1918,  Vol.  23,  pp.  43-47. 

Dysentery  and  Allied  Bacilli.  Journal  of  Infectious  Diseases, 
1920,  Vol.  21,  pp.  31-39. 

A  Facultative  Spore  Forming  Lactose  Fermenting  Organism  from 
Iowa  Surface  Waters.  (B.  macerans.)  Joint  paper  with 
Jack  J.  Hinman,  Jr.  Journal  of  the  American  Water  Works 
Association,  1922,  Vol.  9,  pp.  330-343. 


549742 


NOTES  ON  BACT.  COLI  AND  BACT.  AEROGENES 

MAX  LEVINE, 

From  the  Department  of  Pathology  and  Bacteriology, 
State  University  of  Iowa,       '•'•  < 

Iowa  City,  Iowa. 

Read  before  Laboratory   Section,   American   Public   Health   Association,   at   San   Francisco,    Cal., 

September  16,  1920. 


Accurate  information  on  the  relative  incidence  of  Bact.  coli 
and  Bact.  aerogenes  in  nature  would  aid  materially  in  the  inter- 
pretation of  the  colon  test  in  water  analysis.  Professor  Levine 
suggests  the  lines  along  which  selective  media,  for  the  isolation 
of  these  organisms,  may  be  devised. 


IN  studies  on  the  distribution  of  B. 
coli  and  B.  aerogenes,  the  author  has 
given  preference  to  the  plate  method 
of  isolation.  This,  also,  seems  to  be  the 
view  of  most  other  investigators  who 
have  concerned  themselves  with  similar 
studies.  In  water  analysis,  on  the  other 
hand,  the  plate  method  of  direct  isolation 
is  inconvenient  and  practically  impos- 
sible when  dealing  with  large  samples 
(10  to  100  cc.),  and  preliminary  enrich- 
ment, therefore,  is  resorted  to.  What 
happens  in  the  preliminary  enrichment 
tube  as  to  the  relative  abundance  of  B. 
coli  and  B.  aerogenes,  is  not  definitely 
known  as  far  as  the  author  is  aware. 
Overgrowths  of  one  or  the  other  of  these 
organisms  in  the  preliminary  enrichment 
tube  make  it  extremely  difficult,  if  not 
impossible,  to  correlate  the  water  results 
with  the  findings  that  have  been  reported 
(with  the  plate  method)  on  the  distribu- 
tion of  B.  coli  and  B.  aerogenes  in  nature. 
The  author  has  felt  for  some  time  that 
before  much  light  will  be  thrown  on  the 
true  relative  incidence  of  B.  coli  and  B. 
aerogenes  in  water  and  in  feces,  etc.,  it 
will  be  necessary  first,  so  to  modify  our 
preliminary  enrichment  media,  or  other 
conditions,  as  to  enable  the  investigator 
to  isolate  or  suppress  either  B.  coli  or 
B.  aerogenes  at  will. 

With  this  in  mind,  studies  were  begun 
to   determine   the    influence   of    various 


factors  such  as  dyes,  bile-salts,  concen- 
tration of  peptone,  etc.,  on  the  rate  of 
multiplication  of  B.  coli.  In  general,  it 
was  found — 

(1)  That  B.  coli  would  not  grow  in 
y2  percent  peptone  with  crystal  violent 
in   a  dilution  of   1-200,000,   or  brilliant 
green  in  a  dilution  of  1-1,000,000. 

(2)  That    bile-salts    stimulated    the 
growth  of  B.  coli  when  the  concentration 
was  less  than  0.5  percent,  but  showed  a 
marked  inhibitory  action  if  the  concen- 
tration were  raised  to  0.7  or  1.0  percent. 
It  was  intended  to  continue  this  work 
with  B.  aerogenes,  but  the  outbreak  of 
the  war  interfered  with  the  plans.    The 
following  factors  are  now  being  studied 
as  to  their  influence  on  the  growth  of 
B.  coli  and  B.  aerogenes. 

1 .  Temperature. 

2.  Boric  acid. 

3.  Crystal  violet. 

4.  Brilliant  green. 
Temperature. — That  B.  coli  and  B. 

aerogenes  have  different  optimum  growth 
temperatures  may  be  inferred  from  the 
literature.  Rogers  and  his  associates 
have  often  mentioned  the  necessity  for 
using  a  relatively  low  temperature  (30° 
C.)  for  growth  of  some  strains  of  B. 
aerogenes  isolated  from  grains.  Sim- 
ilarly Rettger  reports  that  in  studying 
the  distribution  of  the  colon  group  in  un- 
polluted soils  a  temperature  of  30°  C. 


Reprinted   from   January,    1921,  issue   of   the  American  Journal    of   Public    Health. 


was  desirable  for  isolation  of  the  B.  acr- 
o genes  types. 

As  to  B.  coli,  a  temperature  of  40°  C. 
has  of!'T  'x>','n  recuii«i'iei.'d'.'d  for  its  iso- 
lation and  in  the  Eijkman  test  46°  C.  is 
employed  for  the  isolation  of  the  organ- 
ism from  water.  In  fact,  it  has  been 
observed  that  the  maximum  rate  of  mul- 
tiplication of  B.  coli  is  at  about  45°  C. 

The  author  observed  that  in  peptone 
lactose  media  at  43°  C.  (in  a  water  bath) 
all  the  cultures  of  B.  coli  (16)  grew 
luxuriantly  as  evidenced  by  strong  tur- 
bidity in  24  hours,  but  69  percent  showed 
no  gas  or  only  a  bubble  in  24  hours.  Of 
20  cultures  of  B.  aerogenes,  on  the  other 
hand,  16  showed  no  growth,  2  slight,  and 
2  grew  luxuriantly. 

Boric  Acid. — In  agar  of  the  follow- 
ing composition:  peptone  1.0  percent, 
agar  1.5  percent,  dipotassium  phosphate 
0.3  percent,  and  glucose  .05  percent  with 
0.63  percent  of  boric  acid,  B.  aerogenes 
failed  to  grow,  whereas  B.  coli  grew  lux- 
uriantly. In  liquid  media,  1.0  percent 
peptone  with  0.63  percent  boric  acid,  B. 
coli  multiplied  slowly,  while  B.  aerogenes 
died  off  as  evidenced  by  the  following 
figures : 

Culture  19b,  B.  coli  increased  from 
65,000  per  c.c.  to  1,500,000  per  c.c.  in  48 
hours,  while  B.  aerogenes  was  reduced 
from  2,300  per  cc.  to  20  in  24  hours  and 
to  0  in  48  hours.  It  was  found  in  subse- 
quent studies,  however,  that  the  differ- 
ence in  concentration  of  boric  acid,  which 
did  not  inhibit  B.  coli  and  which  did  in- 
hibit B.  aerogenes,  was  so  close  that  it 
could  not  be  safely  employed  as  a  se- 
lective agent. 

Crystal  Violet. — One  percent  pep- 
tone water  containing  */2  percent  lactose 
and  varying  concentrations  of  crystal 
violet  were  inoculated  from  48-hour  pep- 
tone cultures  of  B.  coli  and  B.  aerogenes. 
Five  different  strains  of  each  species 
were  employed.  A  concentration  of 
1-100,000  of  crystal  violet  prevented  the 
growth  of  all  the  cultures  of  B.  coli, 
whereas  all  of  the  B.  aerogenes  grew 
heavily.  One  culture  of  B.  coli  failed  to 


grow  in  a  dilution  of  1-250,000  of  crystal 
violet. 

Decreasing  the  concentration  of  pep- 
tone to  y>  percent  increased  markedly  the 
inhibitory  action  of  the  dye.  Thus  in  y2 
percent  peptone  lactose  solution  none  of 
the  B.  aerogenes  grew  with  a  dye  con- 
centration of  1-100,000,  but  all  grew 
luxuriantly  in  1-250,000  crystal  violet. 
Among  the  B.  coli  cultures,  all  were  in- 
hibited in  1-250,000  dilution  of  the  dye 
and  two  failed  to  grow  in  a  dilution  of 
1-500,000. 

Brilliant  Green. — Some  time  ago,  the 
author  was  informed  that  growths  of  B. 
coli  are  rarely  encountered  in  the  isola- 
tion of  B.  typhosns  from  stools  by  the 
use  of  cosine  brilliant  green  agar,  and 
that  if  a  growth  other  than  B.  typhosus 
was  present,  it  was  very  likely  to  be  B. 
aerogenes.  This  suggested  that  the  in- 
hibitory action  of  brilliant  green  was 
much  greater  for  B.  coli  than  for  B. 
aerogenes. 

In  a  medium  consisting  of  1.0  percent 
peptone  and  0.5  percent  lactose  with  vari- 
ous concentrations  of  brilliant  green, 
four  cultures  of  B.  aerogenes  grew  very 
luxuriantly  in  a  concentration  of  1-750,- 
000,  whereas  one  failed  to  grow  in  this 
concentration,  but  grew  very  well  in 
1-1,000,000  dilution  of  the  dye.  The  5 
cultures  of  B.  coli,  on  the  other  hand,  all 
failed  to  grow  in  1-750,000  of  the  dye, 
3  did  not  grow  in  a  dilution  of  1-1,000,- 
000  and  2  failed  to  grow  even  in  a  dilu- 
tion of  1-1,500,000. 

Reducing  the  concentration  of  peptone 
to  J^  percent  increased  very  markedly 
the  antiseptic  action  of  brilliant  green. 
The  five  B.  coli  cultures  now  failed  to 
grow  even  in  a  dilution  of  1-3,000,000. 
The  B.  aerogenes  cultures  grew  luxuri- 
antly in  a  dilution  of  1-2,000,000.  Four 
grew  in  a  dilution  of  1-1,500,000,  but 
only  1  grew  in  more  concentrated  solu- 
tions of  the  dye. 

The  selective  action  of  brilliant  green 
was  even  more  strikingly  shown  by  the 
use  of  a  plate  medium  consisting  of  the 
simplified  eosine  methylene  blue  agar 


with  various  concentrations  of  the  bril- 
liant green.  Four  cultures  of  B.  aero- 
genes  and  five  of  B.  coll  were  employed 
with  the  following  results : 

With  a  dilution  of  1-100,000  of  bril- 
liant green,  none  of  the  B.  coli  grew  at 
all.  All  of  the  B.  aerogencs  grew,  but 
the  colonies  were  only  half  as  large  as 
the  controls  indicating  a  marked  inhibi- 
tion. With  1-200,000  dilution,  B.  coli 
still  failed  to  grow  whereas  B.  aerogenes 
grew  reasonably  well,  but  not  as  lux- 
uriantly as  the  controls.  With  1-300,000 
of  the  dye,  three  of  the  B.  coli  still  failed 
to  show  any  evidence  of  growth  and  two 
others  grew  very  poorly.  All  the  aero- 
genes  showed  a  very  heavy  growth.  With 


1-400,000  brilliant  green,  the  growths  of 
B.  aerogenes  were  as  luxuriant  as  the 
controls,  whereas  2  cultures  of  B.  coli 
failed  to  grow  and  the  three  others 
showed  very  small,  stunted  non-char- 
acteristic colonies. 

In  conclusion,  it  may  be  said  that  these 
preliminary  studies  indicate  that  the  con- 
centration of  peptone  exerts  a  marked 
influence  on  the  inhibitory  action  of  dyes 
in  culture  media,  and  that  it  appears 
feasible  to  devise  both  liquid  and  solid 
media  which  will  inhibit  B.  coli.  but  not 
B.  aerogenes.  The  most  promising  in- 
hibitory agent  which  we  have  as  yet  en- 
countered for  this  purpose  is 'brilliant 
green. 


Acid-Production  and  Other  Characters  of 

Bacillus-Coli-Like  Bacteria  from 

Feces  and  Sewage 


MAX     L  E  V  I  N  E 


Reprinted  from 
THE  JOURNAL  OF  INFECTIOUS  DISEASES,  Vol.  19,  No.  6,  December,  1916,  pp.  773-805 


ACID-PRODUCTION      AND      OTHER      CHARACTERS      OF 
BACILLUS-COLI-LIKE      BACTERIA      FROM 
FECES     AND     SEWAGE  ' 

MAX    LEVINE 

From  the  Department  of  Bacteriology  of  the  Iowa  State  College,  Ames 

The  ability  to  decompose  carbohydrates  with  the  formation  of 
acid  has  long  been  recognized  as  one  of  the  characteristics  of  Bacillus- 
coli-like  bacteria.  This  property  of  acid-production  is  the  basis  for 
the  isolation  of  B.  coli  on  the  Wurtz  litmus-lactose-agar  plate,  and 
also  for  the  separation  of  B.  coli  from  its  relatives,  B.  typhosus  and 
B.  paratyphosus,  on  the  Conradi-Drigalski  agar  medium.  The  ability 
to  ferment  various  substances  has  been  further  utilized  as  a  basis  for 
the  subdivision  of  the  colon-aerogenes  group.  In  these  studies  on 
classification,  however,  attention  has  been  focused  upon  gas-formation 
rather  than  upon  acid-production. 

Browne,1  in  an  extensive  study  of  certain  factors  influencing  acid- 
production,  points  out  that  Bacillus-coli-like  bacteria  isolated  from 
oysters  formed  less  acid  from  carbohydrates  than  those  isolated  from 
human  stools,  and  he  attributed  the  difference  to  a  loss  of  fermenting 
power  by  the  organisms  in  their  passage  through  sewage  from  the 
intestines  to  the  oysters.  Unfortunately,  this  investigator  did  not  dif- 
ferentiate the  different  types  of  organisms  with  which  he  was  work- 
ing. It  is  entirely  probable  that  the  smaller  amount  of  acid  observed 
among  the  oyster  strains  was  due  to  a  greater  incidence  of  some  par- 
ticular type  or  species  of  Bacillus-coli-like  microorganism  rather  than 
to  a  loss  of  fermenting  power  on  the  part  of  the  intestinal  forms. 
[After  the  completion  of  the  experimental  work  for  this  paper,  an 
article  appeared  by  Clark  and  Lubs,2  who  pointed  out  that  bovine  fecal 
strains  of  B.  coli  give  rise  to  a  higher  H+- ion  concentration  in  glu- 
cose than  do  nonfecal  (grain)  strains.] 

The  present  investigation  was  undertaken  to  determine  the  fol- 
lowing : 

1.  Do  Bacillus-coli-like  organisms  from  different  sources  (par- 
ticularly animal  sources)  give  rise  to  different  amounts  of  acid  in  the 

1  Jour.  Infect.  Dis.,  1914,  15,  p.  580. 
3  Ibid.,  1913,  17,  p.  797. 


MAX    LEVINE 

decomposition  of  fermentable  substances,  and  if  they  do,  are  the  dif- 
ferences in  acid-formation  sufficiently  great  to  warrant  quantitative 
acid-production  as  a  reliable  differential  index? 

2.  Is  quantitative  acid-production  correlated  with    (a)    the   Mac- 
Conkey  types,3  (b)  the  Voges-Proskauer  reaction,  or  (c)  gas-forma- 
tion? 

3.  Are  the  morphologic  and  physiologic  characteristics  correlated 
with  the  source? 

CULTURES    STUDIED 

Altogether  167  organisms  were  studied;  156  were  obtained  from 
sewage  and  from  the  feces  of  horse,  cow,  sheep,  pig,  and  man,  and 
11  were  from  the  collection  of  the  American  Museum  of  Natural 
History. 

The  method  of  isolation  has  been  described  in  a  previous  paper.4 
They  were  all  of  the  colon-bacillus  group;  that  is,  gram-negative, 
usually  short  rods,  which  formed  gas  from  glucose  and  lactose,  coagu- 
lated milk,  and  did  not  liquefy  gelatin  in  20  days. 

PREPARATION  OF  MEDIA 

T*he  medium  employed  for  tests  of  acid-production  consisted  of  1%  peptone 
water  to  which  was  added  1%  of  the  test  substance.  Peptone  water,  rather 
than  nutrient  broth,  was  used,  to  eliminate  the  formation  of  acid  from  traces 
of  any  other  fermentable  substance  which  might  be  present  in  beef  extract 
or  meat  infusion.  The  reaction  of  the  medium  was  neutral  to  phenolphthalein. 

Sterilisation. — The  medium  was  tubed  (10  c.c.  in  Durham  fermentation 
tubes)  and  sterilized  in  the  autoclave  for  10  minutes  at  10  pounds  pressure, 
which  is  a  shorter  period  than  is  recommended  in  the  Standard  Methods  for 
Water  Analysis  (1912).  Immediately  on  removal  from  the  autoclave  the  medium 
was  rapidly  cooled  by  immersion  in  cold  water,  then  incubated  for  2  or  3  days 
at  37  C.  in  order  to  eliminate  tubes  which  had  escaped  proper  sterilization. 
Nonsterile  tubes  were  rarely  found.  Sufficient  medium  was  prepared  at  one 
time  to  permit  a  test  of  all  the  cultures  on  one  substance.  Variations  in  the 
composition'  of  the  medium  were  reduced  to  a  minimum  by  using  distilled 
water  and  the  same  bottle  of  Witte's  peptone  throughout  the  work. 

DETERMINATION   OF   ACID-PRODUCTION 

Acid-production  was  determined  in  the  following  manner.  A  tube  of 
peptone  water  was  inoculated  from  an  agar-slant  stock  culture  and  incubated 
at  the  body  temperature  (37  C.)  for  24  hours.  Two  standard  4-mm.  loops 
of  this  24-hour  peptone  culture  were  then  inoculated  into  each  of  2  tubes  of 
peptone  medium  containing  the  test  substance  and  incubated  for  36  hours  at 
37  C.  Acid-production  in  duplicate  tubes  varied  so  little  that  duplicates  were 
not  employed  with  dulcitol,  galactose,  maltose,  glycerol,  and  salicin. 

3  Jour.  Hyg.,  1905,  5,  p.  333;  1909,  9,  p.  86. 

4  Levine:  Jour.  Infect.  Dis.,  1916,  18,  p.  358. 


BACILLUS-COLI-LlKE  BACTERIA   FROM  *FECES  AND   SEWAGE  5 

The  body  temperature  was  selected  for  incubation,  because,  as  was  shown 
by  Browne,1  acid-production  by  B.  coli  is  most  rapid  at  this  temperature.  He 
also  showed  that  with  certain  carbohydrates  and  alcohols  the  maximal  amount 
of  acid  is  formed  in  less  than  24  hours.  Thirty-six  hours'  incubation  was 
employed  for  convenience  in  this  study.  With  the  alcohol,  glycerol,  and  the 
glucosid,  salicin,  the  36-hour  .incubation  period  was  not  sufficient.  Acid-  and 
gas-formation  from  these  substances  were  therefore  determined  after  72 
hours'  growth. 

Titration. — As  the  acidity  of  distilled  water  varied  on  different  days,  the 
following  technic  was  adopted  in  order  to  obviate  tedious  subtractions  of 
checks.  To  a  pail  of  distilled  water  (6  to  8  liters)  was  added  1%  phenol- 
phthalein  solution  (5  gm.  phenolphthalein  in  1  liter  of  50%  alcohol).  The 
water  was  boiled  vigorously  for  15  minutes  and  then  neutralized  with  sodium 
hydroxid.  Of  this  neutral  distilled  water,  containing  the  indicator,  45  c.c.  were 
dipped  out  into  an  evaporating  dish  or  casserole,  5  c.c.  of  the  test  culture  were 
added,  and  the  amount  of  acid  determined  by  titration  with  N/20  NaOH 
without  boiling.  4 

TREATMENT    OF    RESULTS 

A  few  extremely  high  or  low  results  will  influence  considerably 
the  average  acid-production  of  a  collection  of  organisms.  The  use  of 
unqualified  averages  may  therefore  lead  to  a  misconception  of  the 
acid-producing  properties  of  a  group.  To  supplement  the  arithmetic 
mean,  or  numeric  average,  some  statement  should  be  made  as  to  the 
distribution  of  the  variates  about  the  average.  This  may  be  indicated 
by  the  probable  error  or  by  the  standard  deviation.  The  coefficient 
of  variability  (the  ratio  of  the  standard  deviation  to  the  mean)  is  an 
excellent  abstract  measure  of  variability.  The  modal  acid-production 
(the  amount  of  acid  most  frequently  formed)  may,  under  certain  con- 
ditions, be  of  greater  significance  than  the  average  amount  of  acid 
formed. 

In  this  study  the  mean,  the  probable  error  of  a  single  variate,  the 
standard  deviation,  the  coefficient  of  variability,  and  the  empirical 
mode  are  employed. 

The  standard  deviation  is  the  measure  of  variability  most  com- 
monly employed,  particularly  by  mathematicians.  It  may  be  expressed 
mathematically  as 


is  the  number  of  variates, 
or  observations,  "d"  the  deviation  of  the  individual  variates  from  the 
mean,  and  "f"  the  frequency  of  a  deviation  "d".  The  standard  devia- 
tion serves  to  indicate  whether  the  departures  from  the  mean  are 


6  •»  MAX    LEVINE 

small  or  great.    The  closer  the  individual  organisms  group  themselves 
about  the  mean,  or  average,  the  smaller  the  standard  deviation. 

An  example  may  make  clear  the  meaning  and  significance  of  the  standard 
deviation.  Suppose  that  the  amounts  of  acid  formed  by  a  group  (A)  of  4 
organisms  in  glucose  broth  are  2.1,  2.2,  2.2,  and  2.3%  normal  acid,  and  that 
those  formed  by  another  group  (B)  of  4  organisms  are  1.9,  2,  2.4,  and  2.5% 
normal  acid.  The  average  for  each  group  is  2.2,  but  mere  inspection  shows 
that  the  organisms  in  Group  A  and  those  in  Group  B  are  quite  differently 
distributed  with  respect  to  this  average.  In  large  collections  of  data  inspection 
is  impracticable,  but  the  standard  deviation  serves  well  in  its  place.  The 
standard  deviation  in  Group  A  is  ±  0.07  while  for  Group  B  it  is  ±  0.25.  The 
larger  deviation  in  B  denotes  that  the  individuals  in -the  group  wander  farther 
away  from  the  average  than  do  those  in  Group  A. 

The  probable  error  is  employed  to  indicate  what  confidence  is  to 
be  placed  in  statistical  results.  The  reliability  of  the  mean  and  stand- 
ard deviation  may  be  determined  by  calculating  their  probable  errors, 
but  in  this  paper  only  the  probable  error  of  a  single  variate  is  con- 
sidered. In  a  normal  distribution  the  probable  error  of  a  single 
variate  of  a  series  of  observations  is  denned  as  that  departure  from 
the  mean,  on  either  side,  within  which  exactly  one-half  of  the  variates 
are  found ;  that  is,  if  in  the  study  of  acid-production  by  a  large  number 
of  organisms,  it  is  found  that  the  mean  (average)  amount  of  acid 
formed  is  2.25%  normal,  and  that  the  probable  error  of  a  single  obser- 
vation is  ±0.15,  then  half  of  the  organisms  have  formed  between 
2.1%  and  2.4%  normal  acid. 

The  coefficient  of  variability  is  the  ratio  of  the  standard  deviation 
to  the  mean  (^  ).  It  is  an  abstract  measure  of  variability  and  may 
therefore  be  employed  to  advantage  for  comparing  variability  among 
different  characters,  or  in  the  same  character  among  different  groups 
of  organisms,  particularly  if  their  means  differ  widely.5 

ACID-PRODUCTION    IN    SUBSTANCES    FERMENTED    BY    ALL    OF    THE 
TEST  ORGANISMS 

Glucose,  galactose,  mannitol,  maltose,  and  lactose  were  decomposed 
with  gas-production  by  all  strains. 

A.   GLUCOSE 

The  frequency  distributions  of  the  organisms  with  respect  to  acid- 
formation  from  glucose  are  shown  in  Table  1,  where  the  relation  of 

8  For  a  detailed  description  of  these  constants  the  reader  is  referred  to  Principles  of 
Breeding  (1907),  by  E.  Davenport;  Statistical  Methods  (1904),  by  C.  B.  Davenport;  Precision 
of  Measurements  (1909),  by  Goodwin,  and  to  an  Introduction  to  the  Theory  of  Statistics 
(1916),  by  Yule. 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  7 


TABLE     1 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  GLUCOSE 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 

Strains 

Voges- 
Proskauer 

I 

II 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 
2.20-2.39 

3 

4 
8 
28 
10 
1 

4 
3 
5 
8 
6 

3 
9 
3 
10 
17 

1 
3 
2 
1 
3 
6 
8 
8 
2 

2 
5 
10 

2 

1 
3 

1 

1 
5 
11 

3 

5 
2 
9 

1 

5 
2 

7 
8 
9 

1 
4 
14 
5 

1 

1 

3 
11 
6 
13 
5 

1 
3 
2 
11 
19 
22 
54 
41 
3 

1 
3 
1 
9 
15 
20 
54 
41 
3 

1 
2 
4 
2 

Total    acid- 
formers.. 

54 

26 

42 

34 

19 

22 

20 

31 

25 

39 

156 

147 

9 

Mode...  

1.90 

1.90 

2.10 

2.00 

2.10 

2.10 

1.90 

2.10 

1.90 

1.90 

1.90 

1.90 

1.50 

Mean  

1.85 

1.77 

1.84 

1.71 

2.03 

1.76 

1.70 

1.79 

1.91 

1.71 

1.80 

1.82 

1.46 

Probable  error 

±.14 

±.18 

±.19 

±.29 

±.11 

±.32    ±.17 

±.18 

±•11 

±.18 

±.20 

±.20 

±.12 

Standard  devi- 
ation   

±.21 

±.27 

±.28 

±.43 

±.16 

±•47 

±.25 

±.27 

±.17 

±.26 

±.30 

±.30 

±.18 

Coefficient    of 
variation.... 

11.3 

15.2 

15.2 

25.1 

7.9 

26.7 

14.7 

15.1 

8.9 

15.2 

16.6 

16.5 

12.3 

acid-production  to  the  source,  to  the  MacConkey  types,  and  to  the 
Voges-Proskauer  reaction  is  also  indicated. 

The  mode  for  acid-production  by  all  strains  is  at  1.9%  normal, 
with  the  mean  at  1.8%  normal  acid. 

The  means  or  average  quantities  of  acid  formed  by  the  MacConkey 
types  indicate  that  Types  III  (communior)  and  I  (acidi-lactici)  pro- 
duce about  equal  quantities  of  acid  (1.84  and  1.85%  normal  respec- 
tively), while  Type  II  (communis)  forms  somewhat  less  (1.77%), 
and  Type  IV  (aerogenes)  the  least  amount  (1.71%).  A  comparison 
of  Type  IV,  which  forms  the  smallest  quantity  of  acid,  with  Type  I, 
which  gives  the  greatest  amount,  indicates  that  the  means  tend  to  exag- 
gerate the  difference  between  the  two  types  in  ability  to  form  acid 
from  glucose.  Type  I  has  a  well-defined  mode  at  1.90%  and  Type  IV 
has  a  very  indistinct  mode  at  about  the  same  point.  The  standard 
deviation  in  Type  IV  is  ±0.43,  or  three  times  as  great  as  the  difference 
between  the  means  of  the  two  MacConkey  types.  Similar  observations 


MAX    LEVINE 

may  be  made  on  the  other  types.  It  is  therefore  apparent  that  quanti- 
tative acid-production  in  glucose  is  not  a  reliable  criterion  for  differen- 
tiation of  the  MacConkey  types. 

There  are  many  irregularities  in  the  frequency  distributions  of 
organisms  from  different  sources  with  respect  to  acid-production  in 
glucose.  The  organisms  from  horse  and  man  group  themselves  in  a 
manner  simulating  a  normal  distribution,  but  the  frequency  curves  of 
those  from  cow,  sheep,  and  pig,  contain  2  modes.  These  multiple 
modes  are  probably  due  to  the  choice  of  classes  and  to  the  small  num- 
ber of  cultures  from  each  source.  In  the  other  test  substances  mul- 
tiple modes  are  very  infrequent.  In  the  column  headed  "Mode,"  in 
the  frequency  tables,  the  primary  mode  is  recorded. 

The  distribution  of  organisms  from  the  sheep  is  interesting.  Two 
distinct  groups  are  indicated,  one  of  which  generally  produces  more 
than  2%  normal  acid  and  the  other  usually  less  than  1%.  Of  the  5 
low-acid-formers,  4  are  from  a  single  animal  (all  the  cultures  obtained 
from  that  animal),  and  they  are  distinguished  morphologically  from 
all  the  other  sheep  strains  in  that  they  are  distinctly  longer. 

Among  the  sewage  strains  2  well-defined  modes  are  evident,  at 
1.9%  ana  1.5%  normal  acid,  corresponding  with  the  modes  of  the 
Voges-Proskauer-negative  and  the  Voges-Proskauer-positive  organ- 
isms respectively. 

In  a  consideration  of  the  different  animal  sources  it  appears  that 
the  average  amount  of  acid  formed  from  glucose  by  Bacillus-coli-like 
organisms  from  horse  (2.03%)  is  greater  than  the  amounts  formed 
by  strains  from  pig,  sheep  and  cow  (1.70,  1.76,  and  1.79%),  while 
the  amount  formed  by  human  strains  is  intermediate  (1.91%  normal). 
This  relationship  does  not  hold  for  other  test  substances  and  there 
does  not  seem  to  be  any  marked  relation  between  the  quantities  of  acid 
produced  from  glucose,  and  those  formed  from  other  fermentable 
carbohydrates  or  alcohols  by  colon-bacillus-like  organisms  from  the 
animals  recorded  here. 

Quantitative  acid-formation  is  better  correlated  with  the  Voges- 
Proskauer  reaction  than  with  the  source  or  with  MacConkey's  groups. 
The  Voges-Proskauer-negative  organisms  give  an  average  of  1.82% 
normal  acid,  with  a  mode  at  1.90%,  while  the  Voges-Proskauer-posi- 
tive strains  1.46%,  with  a  mode  at  1.50%  normal,  and,  altho  the  dif- 
ference, 0.36,  is  probably  not  sufficient  for  reliable  differentiation,  it 
is  nevertheless  significant,  because  rather  striking  differences  in  acid- 
formation  between  the  Voges-Proskauer-positive  and  the  Voges-Pros- 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE 


kauer-negative  strains  are  observed  with  many  other  test  substances, 
as  maltose,  sucrose,  glycerol,  and  dulcitol. 

B.    GALACTOSE 

The  frequency  distributions  with  respect  to  acid-production  in 
galactose  are  shown  in  Table  2.  Less  acid  is  formed  from  galactose 
than  from  glucose,  and  the  frequency  distributions  are  very  nearly 
normal.  Multiple  modes  are  not  present.  The  average  amount  of 

TABLE     2 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  GALACTOSE 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 

Strains 

Voges- 
Proskauer 

I 

II 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

3 
28 
21 
1 

18 
8 

4 
21 

16 

1 

1 

1 
2 
6 
10 
13 

8 
11 

1 
3 
6 

11 

1 

12 
6 

2 
17 
11 

1 
19 
5 

1 
1 
6 
15 
14 
2 

1 

1 
2 
13 
77 
58 
2 

1 

1 
1 

8 
76 
57 
1 

1 
5 

1 
1 

1 

Total    acid- 
formers  

58 

26 

42 

33 

19 

21 

19 

30 

25 

39 

154 

145 

9 

Mode 

1.30 

1.30     1.30 

1.50 

1.50        1.50 

1.30 

1.30 

1.30 

1.30 

1.30 

1.30 

1.10 

Mean.  . 

1.37 

1.36 

1.37 

1.27 

1.42        1.36 

1.35 

1.36     1.33 

1.34 

1.35 

1.36 

1.21 

Probable  error 

-+-.08 

-+-.06 

-+-.09 

-+-.18 

-K07  i    -+-.12 

+.07 

-+-.08    -+-.06 

-+-.14 

-+-.12 

-+-.11 

-+-.16 

Standard  devi- 
ation   

±.12 

±.09 

±.13 

±.27 

±.10 

±.18 

±.11 

±.12 

±.09 

±.20 

±.17 

±.16 

±.23 

Coefficient    of 
variation  — 

8.8 

6.6 

9.5 

21.2 

7.1 

13.2 

8.2 

8.8 

6.8 

14.9 

12.6 

11.8 

19.0 

acid  formed  by  all  strains  is  1.35%  normal,  with  a  distinct  mode  at 
1.30%.  (Acid  was  not  determined  from  2  cultures,  1  from  pig  and 
1  from  sheep,  which  broke  just  before  titration.) 

The  MacConkey  types,  I,  II,  and  III,  each  have  a  mode  at  1.30% 
normal  acid,  and  means  at  1.37,  1.36,  and  1.37%  normal  acid  respec- 
tively. Altho  the  mode  for  Type  IV  is  1.50%  normal  acid — some- 
what higher  than  for  the  other  types — the  mean,  1.27%,  is  lower,  a 
circumstance  indicating  a  greater  variability  in  Type  IV.  This  greater 


10  MAX    LEVINE 

variability  is  indicated  by  the  much  larger  standard  deviation  and 
coefficient  of  variability.  MacConkey  types,  therefore,  cannot  be  dif- 
ferentiated on  the  basis  of  quantitative  acid-production  in  galactose 
as  indicated  by  titration  with  phenolphthalein. 

There  does  not  seem  to  be  any  correlation  between  the  amount  of 
acid  formed  from  galactose  and  the  source  of  the  organisms.  One 
organism  from  the  cow  formed  less  than  0.4%  acid.  It  was  omitted 
in  calculating  acid-production  by  the  group.  If  included,  the  mean  for 
the  cow  strains  becomes  1.30%,  with  a  coefficient  of  variability  of 
19.2%. 

The  Voges-Proskauer-positive  strains  form  somewhat  less  acid 
(1.21%)  than  do  the  Voges-Proskauer-negative  strains  (1.36%).  The 
difference  (0.15%  normal)  is  slight,  but  it  is  greater  than  the  differ- 
ences observed  with  the  MacConkey  types  or  with  the  strains  from 
different  sources.  The  difference  is  of  some  interest,  moreover;  for, 
as  will  appear  later,  whereas  the  Voges-Proskauer-positive  strains 
form  less  acid  from  the  monosaccharids  than  do  the  Voges-Proskauer- 
negative  strains,  the  reverse  is  true  when  more  complex  substances 
(except  lactose)  are  fermented. 

C.    MANNITOL 

The  hexite,  mannitol,  is  attacked  about  as  readily  as  galactose. 
The  average  amount  of  acid  formed  by  all  strains  is  1.32%,  with  a 
sharp  mode  at  1.30%  normal.  (Acid-production  was  not  determined 
in  2  cultures,  1  from  horse  and  1  from  man.)  The  frequency  dis- 
tributions and  the  relation  of  the  Voges-Proskauer  reaction,  the 
source,  and  the  MacConkey  types  to  the  amount  of  acid  formed  from 
mannitol  are  shown  in  Table  3. 

The  mode  for  each  of  the  MacConkey  types  is  at  1.30%,  and  the 
means  are  1.32,  1.30,  1.33,  and  1.31%  normal  acid  respectively.  The 
MacConkey  types  are  therefore  indistinguishable  on  the  basis  of  quan- 
titative acid-production  from  mannitol. 

A  comparison  of  the  amounts  of  acid  formed  by  Voges-Proskauer- 
negative  and  Voges-Proskauer-positive  strains  indicates  that  the  latter 
attack  mannitol  somewhat  more  readily,  but  the  difference  is  not 
appreciable. 

Except  for  the  horse  strains,  which  have  a  mode  at  1.50%,  the 
organisms  from  all  the  other  sources  group  themselves  around  1.30% 
normal  acid  as  a  mode.  In  general,  the  differences  observed  between 
the  means  are  too  slight  to  be  of  any  significance.  The  average  of  the 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE 


11 


sheep  strains  is  the  lowest,  1.21%,  as  compared  with  1.34%  for  human 
strains,  1.38%  for  horse,  1.36%  for  sewage,  1.31%  for  cow,  and  1.28% 
normal  for  pig  strains.  The  lower  average  of  the  sheep  strains  is  due 
to  the  presence  among  them  of  a  few  low-acid-producing  organisms 
rather  than  to  a  lesser  ability  of  the  group  as  a  whole  to  attack  manni- 
tol.  That  sheep  strains  form  acid  from  mannitol  as  readily  as  do  those 
from  the  cow,  pig,  and  man  is  indicated  by  the  coincidence  of  their 

TABLE     3 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN   MANNITOL 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 

Strains 

Voges- 
Proskauer 

I 

II 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

9 

29 
13 

1 

6 
14 
6 

7 
20 
14 

2 
4 

15 
12 

1 

1 
1 
6 
10 

2 
3 
3 
9 
5 

5 
9 
6 

1 
6 
19 
5 

3 
13 

8 

4 

22 
11 
1 
1 

2 
5 

22 
78 
45 
1 
1 

2 
5 
22 

75 
40 

1 

3 
5 

1 

Total    acid- 
formers  

53 

26 

41 

34 

18 

22 

20 

31 

.24 

39 

154 

145 

9 

Mode  

1.30 

1.30 

1.30 

1.30 

1.50 

1.30 

1.30 

1.30 

1.30 

1.30 

1.30 

1.30 

1.50 

Mean  

l.S2r 

1.30 

1.33 

1.31 

1.38 

1.21 

1.31 

1.28 

1.34 

1.36 

1.32 

1.31 

1.48 

Probable  error 

±.10 

±.09 

±.09 

±.17 

±.11 

±.18 

±.10 

±.09 

±.09 

±.12 

±.12 

±.12 

±.12 

Standard  devi- 
ation   

±.15 

±•14 

±.14 

±.26 

±.17 

±.26 

±.15 

±.14 

±.13 

±.18 

±.18 

+.18 

±.18 

Coefficient    of 
variation  — 

11.3 

10.8 

10.5 

19.8 

12.3 

21.2 

11.4 

10.9 

9.7 

13.2 

13.6 

13.7 

12.1 

modes.  The  strains  from  the  horse  tend  to  form  somewhat  more 
acid  than  do  those  from  other  animals,  but  the  difference  is  too  slight 
to  be  of  any  differential  significance. 

D.  LACTOSE 

The  amount  of  acid  formed  from  the  disaccharid,  lactose,  in  1  % 
lactose  peptone  solution  is  very  nearly  the  same  as  that  formed  from 
the  monosaccharid,  galactose,  and  from  the  hexite,  mannitol.  The 
mode  for  all  strains  is  at  1.30%,  and  the  mean  is  also  at  1.30%  normal 
acid.  The  frequency  distributions  are  shown  in  Table  4. 


12 


MAX    LEVINE 


MacConkey  Type  II  has  an  ill-defined  mode  at  1.50%,  and  the 
mean  is  1.25%  normal  acid.  The  mode  for  the  other  types  (I,  III, 
IV)  is  at  1.30%,  and  the  means  are  1.31,  1.32,  and  1.33%  normal  acid 
respectively.  The  MacConkey  types  are  indistinguishable  on  the  basis 
of  quantitative  acid-production  in  lactose. 

There  is  no  evident  relation  between  the  source  and  the  amount 
of  acid  produced  from  lactose. 

TABLE     4 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  LACTOSE 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

.Source 

All 
Strains 

Voges- 
Proskauer 

I 

11 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
i.00-1.19 
1.20-1.39 
1.40-1.69 
1.60-1.79 
1.80-1.99 
2.00-2.19 

1 
13 
30 
8 

2 

1 

3 

1 
1 
9 
11 

11 
19 
10 

2 

42 

6 

15 
10 
3 

4 
10 
5 

5 
1 
3 
9 
4 

1 

3 
1 
1 
11 
3 

6 
18 

7 

12 
11 
2 

2 
1 
20 
13 
1 
2 

1 

3 
8 
25 
73 
39 
5 
2 

1 

3 

7 
24 
67 
38 
5 
2 

1 
1 
6 
1 

Total    acid- 
formers  

54 

26 

34 

19 

22 

20 

31 

25 

39 

156 

147 

9 
1.30 

Mode  

1.30 

1.50 

1.30 

1.30 

1.30 

1.50 

1.30 

1.30 

1.30 

1.30 

1.30 

1.30 

Mean    

1.30 

1.25 

1.31 

1.32 

1.31 

1.25 

1.16 

1.81 

1.22 

1.38 

1.30 

1.31 

1.26 
±.11 

Probable  error 

±.12 

±.22 

±.11 

±.16 

±.09 

±.19 

±.22 

±.09 

±.09 

±.13 

±.15 

±.15 

Standard  devi- 
ation      .  ... 

±.17 

±.32 

±.17 

±.23 

±.14 

±.28 

±.33 

±.13 

±.13 

±.20 

±.22 

±.22 

±.16 

Coefficient    of 
variation  — 

13.1 

25.6 

13.0 

17.4 

10.7 

22.4 

28.4 

10.0 

10.6 

14.5 

16.9 

16.8 

12.7 

There  is  no  distinction  between  the  amounts  of  acid  produced 
from  lactose  by  Voges-Proskauer-positive  and  Voges-Proskauer-nega- 
tive  strains.  Both  groups  have  their  modes  at  1.30%,  and  the  means 
are  but  slightly  removed  from  the  modes,  being  1.26  and  1.31%  normal 
acid  respectively. 

E.   MALTOSE 

Decomposition  of  the  disaccharid,  maltose,  yields  considerably  less 
acid  than  the  decomposition  of  the  monosaccharids,  glucose  and  galac- 
tose,  the  hexite,  mannitol,  or  the  disaccharid,  lactose,  mentioned.  All 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  13 


strains  considered,  the  average  acid-production  was  0.77%,  with  a 
very  distinct  mode  at  0.7%  normal  acid.  The  frequency  distributions 
of  the  organisms  from  different  sources,  of  the  MacConkey  types,  and 
of  the  Voges-Proskauer  reaction  with  respect  to  acid-production  in 
maltose  are  shown  in  Table  5.  One  organism  apparently  fails  to  show 
acid  but  forms  gas.  This  neutral  reaction  is  presumed  to  be  due  to 
reversion. 

TABLE     5 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  MALTOSE 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 
Strains 

Voges- 
Proskauer 

I 

II 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

7 
31 
10 
6 

1 

2 
17 
6 

6 

24 
8 
2 
2 

1 
18 
9 
4 
2 

9 
9 
1 

3 

19 

13 
3 
4 

3 

15 
12 
1 

6 

18 

1 

1 

4 
16 

8 
6 
4 

1 

16 
90 
33 
12 

4 

1 

16 
90 
31 
.      8 
1 

2 
4 
3 

9 
1.10 

Total    acid- 
formers  

54 
0.70 

25 

42 

34 

19 

22 

20        31 

25 

38 

155 

146 

Mode  

0.70 

0.70 

0.70 

0.80 

0.70 

0.70 

0.70 

0.70 

0.70 

0.70 

0.70 

Mean  

0.76 

0.73 

0.76 

0.83       0.81 

0.67 

0.81     0.77 

0.67 

0.85 

0.77 

0.75 

1.12 

Probable  erroi 

±.11 
±.16 

±.07 

±.13 

±.13 

±.08 

±.05 

±.11 

±.09 

±.08 

±.15 

±•11 

±.09 

±.10 

Standard  devi- 
ation   

±.11 

±.19 

±.19 

±.11 

±.07 

±.16 

±.14 

±.12 

±.23 

±.17 

±.14 

±,15 

Coefficient    of 
variation  — 

21.0 

15.1 

25.0 

22.9 

13.6 

10.4 

19.8 

18.2 

17.9 

27.0 

22.1 

18.7 

13.4 

Each  of  the  MacConkey  types  has  a  sharply  defined  mode  at  0.70% 
normal.  The  means  for  the  types  are  0.76,  0.73,  0.76,  and  0.83% 
normal  acid  respectively.  There  is  no  correlation  between  quantitative 
acid-formation  from  maltose  and  the  MacConkey  types. 

The  relation  of  acid-formation  from  maltose  to  the  source  of 
Bacillus-coli-like  organisms  is  not  at  all  striking.  The  mode  for  each 
source  is  at  0.7%  normal  acid.  The  trend  of  the  variation  among  the 
strains  from  horse,  cow,  pig,  and  sewage,  is  beyond  the  mode,  so 
that  the  means  are  0.81,  0.81,  0.77,  and  0.83%  normal  acid  respec- 


14  MAX    LEVINE 

tively,  while  the  strains  from  man  and  sheep  vary  in  the  other  direc- 
tion, lowering  the  averages  to  0.67%  for  man  and  0.68%  for  sheep. 
It  should  be  pointed  out  that  the  relatively  high  average  for  sewage, 
0.83%,  is  due  to  the  presence  of  Voges-Proskauer-positive  organisms. 
The  average  for  the  sewage  strains  exclusive  of  the  Voges-Proskauer- 
positive  organisms,  is  0.73%  normal  acid.  Acid-production  in  maltose 
can  not  be  considered  a  reliable  index  for  differentiation  of  Bacillus- 
coli-like  organisms  from  the  sources  studied. 

There  is  a  rather  marked  and  distinct  relation  between  quantitative 
acid-production  in  maltose  and  the  Voges-Proskauer  reaction.  It 
appears  from  Table  5  that  the  Voges-Proskauer-negative  strains  occa- 
sionally form  more  than  1%  acid,  but  usually  less  than  0.8%,  while 
the  Voges-Proskauer-positive  strains  usually  form  more  than  1% 
and  never  less  than  0.8%  normal  acid.  The  mode  for  the  Voges- 
Proskauer-negative  strains  is  at  0.70%  and  the  mean  at  0.78%  normal 
acid.  The  mode  and  mean  for  the  Voges-Proskauer-positive  strains 
are  1.10  and  1.12%  respectively. 

ACID-PRODUCTION    IN    SUBSTANCES    NOT    FERMENTED    BY    ALL 
THE   TEST   ORGANISMS 

The  disaccharid,  sucrose,  the  trisaccharid,  raffinose,  the  glucosid, 
salicin,  and  the  alcohols,  glycerol  and  dulcitol,  were  attacked  by  many, 
but  not  by  all,  of  the  organisms  studied. 

In  calculating  means  and  other  constants  for  acid- formation,  only 
those  organisms  which  attacked  the  test  substances  were  included. 
The  line  of  demarcation  for  acid-production  was  selected  at  0.4% 
normal  acid,  because  in  sucrose,  raffinose,  and  dulcitol  organisms  which 
formed  less  than  this  amount  in  36  hours  at  37  C.  rarely,  if  ever, 
formed  gas,  while  those  which  produced  more  than  0.4%  acid,  prac- 
tically always  formed  gas  also. 

A.  SUCROSE 

In  Table  6  are  shown  the  frequency  distributions  of  acid-produc- 
tion in  sucrose  in  relation  to  the  MacConkey  types,  the  source,  and  the 
Voges-Proskauer  reaction.  The  relation  of  acid-production  to  gas- 
formation,  and  to  the  Voges-Proskauer  reaction,  is  indicated,  also, 
in  Plot  1.  (One  organism,  which  was  overrun  in  titration,  is  not 
included  in  the  calculation.)  Three  modes  are  evident.  One  mode 
is  at  0.1%  normal  acid  and  represents  those  organisms  which  do  not 
form  gas  from  sucrose.  Acid-formation  and  gas-production  in  sue- 


OJ    0.3    0.5  0.7    0.9    //     /.3    /.S    /.7    /.?    2J 


feo/i  SvceosE 


16 


MAX    LEVINE 


rose  are  well  correlated.  Colon-bacilli-like  organisms  which  form  gas, 
also  give  rise  to  acid  and  vice  versa.  Among  the  gas-formers  2  groups 
are  apparent;  one  forms  acetylmethylcarbinol  from  glucose  (V.P.+) 
and  a  relatively  large  amount  of  acid  from  sucrose  (mode  at  1.50% 
normal),  while  the  other  does  not  form  acetylmethylcarbinol  from 
glucose  (V.P. — )  and  gives  rise  to  a  much  smaller  quantity  of  acid 
from  sucrose  (an  extremely  sharp  and  distinct  mode  at  0.70% 
normal). 

MacConkey  Types  I  and  II  do  not  form  acid  from  sucrose.  That 
Types  III  and  IV  are  indistinguishable  on  the  basis  of  quantitative 
acid-production  in  sucrose,  is  apparent  from  Table  6. 

Ten  of  the  organisms  from  cow,  15  from  horse,  20  from  sheep,  10 
from  pig,  and  only  3  from  man,  ferment  sucrose.  The  amount  of  acid 
formed  bears  no  definite  relation  to  the  animal  source,  but  it  should 

TABLE     6 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  SUCROSE 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 
Strains 

Voges- 
Proskauer 

I 

11 

III       IV 

Horse 

Sheep 

Cow 

Pig 

Man!  Sewage 

Neg- 
ative 

Posi- 
tive 

2 
4 
2 

1 
9 

0.00  0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

53 
1 

26 

3 
30 
4 

1 
3 
1 

5 
18 
2 

2 
4 
1 

1 

4 

10 
2 

1 

2 

1 

3 

16 
1 

10 

0 

1 

21 

2 
7 
1 

21 

1 

3 

22 

3 
3 
1 

2 
5 
2 

1 

79 
1 
8 
48 
6 

3 

7 
2 

1 

79 
1 
8 
48 
6 

1 
3 

Total    acid- 
formers 

42 

33 

15 

20 

10 

10 

3 

17 

75 

66 

Mode 

0.70  |  0.70 

0.70 

0.70 

0.70 

0.70 

0.70 

1.50 

0.70 

0.70 
0.74 

1.50 
1.57 

Mean  

0.80     0.91 

0.93 

0.68 

0.72 

0.68 

0.70 

1.18 

0.84 

Probable  error 

±.18 

±.27 

±.20 

±.06 

±.04 

±.07 

±.33 

±.23 

±•14 

±.16 

Standard  devi- 
ation   



-+-.27 

±.40 

±.29 

±.09 

±.06 

±•11 

±.49 

±.34 

±.20 

±.23 
14.6 

Coefficient    of 
variation 

33.8 

44.0 

31.2 

13.2 

8.3 

16.2 

41.5 

40.5 

27.0 

be  noted  that  a  few  cultures  among  the  horse  strains  form  considerably 
more  acid  than  any  of  the  other  animal  strains.    The  high  average  for 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE 


17 


the  horse  strains  is  due  to  the  influence  of  these  few  cultures  and  is 
not  a  characteristic  of  horse  strains  in  general. 

The  high  average,  1.18%  normal  acid,  of  the  17  sewage  strains 
which  attacked  sucrose,  is  due  entirely  to  the  presence  among  them 
of  9  Voges-Proskauer-positive  organisms.  The  mean  for  the  other 
8- sewage  strains  is  0.75%  normal  acid. 

Voges-Proskauer-negative  strains  attack  sucrose  less  readily  than 
the  Voges-Proskauer-positive  strains.  The  means  for  the  two  groups 
are  0.74  and  1.57%,  and  the  empirical  modes  0.7  and  1.5%  normal  acid 
respectively. 

B.    RAFFINOSE 

The  frequency  distributions  of  the  organisms  with  respect  to  acid- 
production  in  raffinose  and  the  relation  of  acid-formation  to  the  Mac- 
Conkey  types,  to  the  source,  and  to  the  Voges-Proskauer  reaction,  are 
given  in  Table  7. 

TABLE     7 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN    RAFFINOSE 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 

Strains 

Voges- 
Proskauer 

I 

II 

III 

2 
13 
10 
8 
8 
1 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

50 
1 

2 
1 

23 
1 

1 

1 

2 

1 
5 
5 
11 
10 
2 

3 

1 
5 
3 
4 
3 

1 
4 
3 
8 
5 
1 

8 
1 

2 
6 
1 
2 

20 

1 
"      5 

2 
2 
1 

21 

2 
1 
1 

21 

1 
2 
2 
4 
7 
2 

73 
1 

18 
18 
20 

1 

73 
1 
4 
18 
18 
19 
13 
1 

1 

6 
2 

Total    acid- 
formers  

4 

42 

34 

16 

22 

11 

U 

4 

18 

82 

73 

9 

Mode 

0.70 

1.10 

0.70 

1.10 

0.90 

0.70 

0.90 

1.30 

1.10 

1.10 

1.30 

0.95 

108 

0.94 

1.03 

0.97 

0.85 

1.05 

1.11 

1.00 

0.96 

1.32 

Probable  error 

±.17 

±.17 

±.15 

±.17 

±.12 
±.18 

±.15 

±•11 

±.19 

±.17 

±.16 

±.08 

Standard  devi- 
ation   

±.25 

±.25 

±.22 

±.25 

±.22 

±.17 

±.28 

±.26 

±.24 

±•11 

Coefficient    of 
variation 

26.3 

22.9 

23.4 

24.3 

18.6 

25.9 

16.2 

25.2 

26.0 

25.5 

8.3 

All  the  strains  considered,  somewhat  more  acid  is  formed  from 
raffinose  (1%  normal)  than  from  sucrose  (0.7%).     No  distinct  mode 


18  MAX    LEVINE 

is  present,  and  the  dispersion  of  the  distribution  is  very  great,  as 
indicated  by  a  large  coefficient  of  variability  (26%). 

MacConkey  Types  I  and  II  generally  do  not  attack  raffinose.  The 
few  strains  in  these  types  which  do,  are  not  sufficient  for  comparative 
purposes.  Type  IV  tends  to  form  more  acid  than  Type  III,  but  the 
difference  is  not  considered  a  reliable  index  for  differentiation. 

There  is  no  apparent  relation  between  the  animal  source  and  the 
amount  of  acid  formed  from  raffinose.  The  mean  for  the  sewage 
strains,  1.11%  normal  acid,  is  higher  than  for  those  from  other 
sources.  This  difference,  as  was  observed  in  the  case  of  sucrose,  is 
due  to  the  presence  of  the  Voges-Proskauer-positive  group  among  the 
sewage  strains.  The  mean  for  the  Voges-Proskauer-negative  strains 
in  sewage  is  0.94%  normal  acid. 

The  Voges-Proskauer-negative  strains  attack  raffinose  less  readily 
than  do  the  Voges-Proskauer-positive  strains.  The  means  for  the  two 
groups  are  0.96  and  1.32%  normal  acid  respectively,  but  the  variability 
among  the  strains  is  such  as  to  make  the  difference  (0.36)  of  question- 
able significance. 

C.  GLYCEROL 

The  alcohol,  glycerol,  is  attacked  by  many  strains  which  form  acid 
but  not  gas.  For  calculating  acid-production  all  strains  which  form 
more  than  0.4%  normal  acid  in  72  hours  at  37  C.  are  regarded  as  acid- 
formers.  The  frequency  distributions  and  the  relation  of  quantitative 
acid-production  to  the  MacConkey  types,  to  the  source,  and  to  the 
Voges-Proskauer  reaction  are  shown  in  Table  8.  A  sharp  mode  is 
observed  at  0.70%  and  the  mean  for  all  the  strains  is  at  0.73%  nor- 
mal acid. 

The  MacConkey  types  are  indistinguishable  on  the  basis  of  quan- 
titative acid-production  in  1%  glycerol  peptone  solution  altho  Type  III 
tends  to  form  somewhat  less  acid  than  the  others. 

The  differences  between  the  means  of  organisms  from  the  various 
animal  sources  cannot  be  regarded  as  significant.  The  sheep  strains 
form  the  least  amount  of  acid  (0.60%-  normal),  while  the  means  for 
strains  from  other  sources  are,  horse  0.67%,  cow  0.71%,  man  0.71%, 
and  pig  0.76%  normal  acid.  The  somewhat  higher  mean  of  the  sew- 
age strains  (0.83%)  is  due,  again,  to  the  influence  of  Voges-Pros- 
kauer-positive strains.  These  eliminated,  the  mean  for  the  Voges- 
Proskauer-negative  strains  in  sewage  is  0.70%  normal. 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE 


19 


One  of  the  Voges-Proskauer-positive  strains  does  not  attack 
glycerol.  This  is  probably  B.  cloacae.  Those  Voges-Proskauer-posi- 
tive organisms  which  do  ferment  glycerol,  generally  form  much  more 
acid  than  the  Voges-Proskauer-negative  fermenting  strains.  The  mean 
for  the  Voges-Proskauer-negative  organisms  coincides  with  the.  mode 
at  0.70%  normal.  The  Voges-Proskauer-positive  organisms  have  a 
mode  at  1.50%,  with  a  mean,at  1.28%  normal  acid. 

TABLE     8 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  GLYCEROL 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 
Strains 

Voges- 
Proskauer 

I 

II 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

2 
10 
25 
16 
1 

1 

1 
16 
8 

1 
2 
20 
13 
3 

3 

•> 
1 
8 
12 
6 
1 
2 
1 

1 

8 
5 
5 

2 
10 
9 

2 
15 
3 

2 
4 
13 
12 

7 
13 
3 
1 
1 

3 
1 
8 
11 
10 
1 
1 
4 

4 
5 

39 
66 
33 
2 
2 
4 

3 
5 
39 
66 
31 
1 
1 

1 

o 
I 
1 

4 

Total    acid- 
formers  

52 
0.70 

25 

39 

30 

18 

19 

20 

29 

25 

35 

146 

138 

s 

Mode  

0.70 

0.50 

0.70 

0.50 

0.50 

0.70 

0.70 

0.70 

0.70 

0.70 

0.70 

1.50 

Mean.. 

0.73 

0.76 

0.67 

0.77 

0.67 

0.60 

0.71 

0.76 

0.71 

0.83 

0.73 

0.70 

1.2a 

Probable  error 

±.10 

±.07 

±•17 

±.17 

±•11 

±.06 

±.07 

±.09 

±.13 

±.20 

±.14 

+.11 

-K16 

Standard  devi 
ation 

±.15 

±.10 

±.25 

±.25 

±.17 

±.09 

±.10 

±.14 

±.19 

±.30 

±.21 

±.16 

±.24 

Coefficient    of 
variation  

20.5 

13.2 

37.4 

32.5 

25.4 

15.0 

14.1 

18.4 

26.8 

36.2 

28.8 

24.3 

16.0 

D.  DULCITOL 

In  Table  9  is  indicated  the  relation  of  the  MacConkey  types,  the 
source,  and  the  Voges-Proskauer  reaction  to  acid-production  in  dul- 
citol. 

MacConkey  Types  I  and  IV  do  not  form  acid  from  dulcitol.  Types 
II  and  III  produce  about  equal  quantities,  0.88%  and  0.81%  normal 
acid  respectively,  but  Type  II  shows  a  greater  variability. 

The  sheep,  pig,  human,  and  horse  strains  attack  dulcitol  more 
vigorously  than  do  the  organisms  from  the  cow.  The  averages  are, 


20 


MAX    LEVINE 


TABLE     9 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN   DULCITOL 


Frequencies 


Percentage 
of                 MacConkey  Type 
Normal 

Arid 

Source 

All 
Strains 

Voges- 
Proskauer 

|      I 

II 

III 

IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

0.00-0.19             53 
0.20-0.39 
0.40-0.59               1 
0.60-0.79 
0.80  0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

1 
5 
7 
6 
6 

1 

16 
21 

I 
3 

33 
1 

6 

2 

5 
6 

11 

1 
1 

9 

9 
1 
2 
5 
3 

18 

4 
4 
5 

19 

1 

2 

1 

2 

23 

2 
6 
4 

3 

86 
3 
6 
23 

27 

3 

80 
3 
6 
23 

27 

7 

6 
3 

Total    acid- 
formers   .      !       1 

24 

41 

11 

11 

10 

13 

6 

15 

66 

63 

3 
1.30 

Mode 

0.70     0.90 

0.90 

0.90 

0.70 

1.10 

0.70 

0.90 

0.90 

Mean 

0.81 

0.88 

0.81 

0.84 

0.72 

0.92 

0.83 

0.85 

0.83 

0.81 
±.11 

1.30 

Probable  error 

±.15    ±.13 

±.07 

±.08 

±.09 

±.11 

±.15 

±.18 

±.15 

Standard  devi- 
ation   

±.22 

±.20 

±.10 

±.12 

±.14 

±.16 

±.22 

±.26 

±.22 

±•17 

Coefficient    of 
variation  

27.2 

22.7 

12.3 

14.3 

19.4 

17.4 

26.5 

30.6 

26.5 

21.0 

pig  0.92%,  sheep  0.84%,  human  0.83%,  and  horse  0.81%,  as  compared 
with  0.72%  normal  acid  for  cow.  The  number  of  fermenting  strains 
from  the  different  sources  is  too  small  for  reliable  comparison  and 
the  differences  here  indicated  are  insignificant. 

Only  3  of  the  Voges-Prpskauer-positive  strains  ferment  dulcitol, 
but  the  amount  of  acid  produced  by  each  of  these  three  organisms 
is  greater  than  that  formed  by  any  of  the  Voges-Proskauer-negative 
strains.  The  mean  for  the  Voges-Proskauer-positive  organisms  is 
1.30%,  and  for  the  Voges-Proskauer-negative  cultures  0.81%  normal 
acid. 

E.  SALICIN 

Acid-production  in  salicin,  as  in  glycerol,  is  not  always  accom- 
panied by  gas-formation.  The  frequency  distribution  with  respect  to 
source,  MacConkey  type,  and  Voges-Proskauer  reaction  is  indicated 
in  Table  10. 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  21 


TABLE     10 

RELATION  OF  SOURCE,  MACCONKEY  TYPE,  AND  VOGES-PROSKAUER  REACTION  TO  ACID-PRODUCTION 

IN  SALICIN 


Percentage 
of 
Normal 
Acid 

Frequencies 

MacConkey  Type 

Source 

All 
Strains 

Voges- 
Proskauer 

I         II 

III  ;    IV 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

Neg- 
ative 

Posi- 
tive 

1 

2 
2 
3 
1 

0.00-0.19 
0.20-0.39 
0.40-0.59 
0.60-0.79 
0.80-0.99 
1.00-1.19 
1.20-1.39 
1.40-1.59 
1.60-1.79 
1.80-1.99 
2.00-2.19 

26 

1 

6 
8 
7 
6 

1 

5 

9 
9 
2 

8 

10 
16 
4 
1 
2 

1 

8 
1 
3 
4 
7 
5 
4 
1 
1 

6 

3 

5 
2 
3 

7 

6 
6 
3 

1 
1 

3 

10 
3 
2 

8 

2 
5 

7 
7 
2 

13 

1 
4 
5 
2 

8 

1 
4 
7 
8 
6 
3 
1 
1 

43 
1 
4 
25 
40 
25 
13 
3 
1 
1 

43 
1 
3 
25 
40 
23 
11 

1 

Total    acid- 
formers  

28 

25 

34 
0.90 

25           13 

15 

18 

23 

12 

31          112  !    103 

9 
1.50 
1.28 

Mode  

0.90 

1.00 

0.90        0.00 

0.90 

0.90 

1.00 

0.90 

1.10 

0.90 

0.90 

Mean  

0.98     0.96 

0.94      0.98 

0.98 

0.94 

0.94 

0.92 

0.83 

1.11 

0.96 

0.94 

Probable  error 

±.16    ±.12 

±.17 

±.21 

±.15 

±.07 

±.12 

±.15 

±.12 

±.21 

±.16 

±.15 

±.22 

Standard  devi- 
ation   

±.23 

±.17 

±.26 

±.30 

±.22 

±•11 

±.17 

±.22 

±.17 

±.31 

±.23 

±.22 

±.33 

Coefficient    of 
variation...  . 

i            | 
23.5    117.7     27.7     30.6 

22.5 

11.7 

18.1 

23.9 

20.5 

27.9 

23.9 

23.4 

25.8 

Inspection  of  Table  10  shows  that  the  MacConkey  types  cannot  be 
differentiated  on  the  basis  of  the  amount  of  acid  formed  from  salicin. 
Neither  is  quantitative  acid-production  an  index  to  the  animal  source. 
The  Voges-Proskauer-positive  strains  give  more  acid  (1.28%  normal) 
than  the  Voges-Proskauer-negative  strains  (0.94%  normal),  but  the 
difference  is  not  as  marked  or  distinct  as  with  sucrose  and  should  not 
be  regarded  as  a  differential  index. 

RESUME 

A  study  of  the  quantities  of  acid  formed  by  Bacillus-coli-like  organ- 
isms from  different  sources  (pig,  cow,  sheep,  horse,  man,  and  sewage) 
when  they  are  inoculated  into  peptone  water  containing  1%  of  various 
fermentable  substances,  indicates  the  following: 

1.  The  MacConkey  types  are  indistinguishable  on  the  basis  of 
quantitative  acid-production  in  the  fermentable  carbohydrates,  the 
alcohols,  and  the  glucosid  studied.  This  is  shown  in  Plot  II,  in  which 


MAX    LEVINE 


1.0 


0.8 


0.6 


the  curves  for  the  different  types  run  almost  parallel  and  very  close 
together. 

2.  That  there  is  no  correlation  between  the  amount  of  acid  formed 
from  the  carbohydrates,  the  alcohols,  and  the  glucosid  studied,  and  the 
animal  source,  is  apparent  from  Plot  III,  in  which  a  parallelism  simi- 
lar to  that  in  Plot  II  is  observed.     The  high  average  of  acid-forma- 
tion in  sucrose  among  the  horse  strains  is  due  to  the  presence  among 
them  of  a  few  high-acid-producing  cultures,  not  to  any  ability  of  horse 
strains,  as  a  whole,  to  yield  more  acid  from  sucrose.     This  has  been 
previously  indicated  in  Table  6. 

3.  In  a  general  way,  the  Voges-Proskauer-positive  strains  isolated 
from  sewage  form  less  acid  from  the  monosaccharids,  but  more  acid 
from  the  more  complex  carbohydrates,  etc.,  (except  lactose)  than  the 
Voges-Proskauer-negative  strains.    That  this  difference  is  not  peculiar 
to  the  strains  isolated  for  this  study,  but  is  characteristic  of  the  Voges- 
Proskauer-positive  and  -negative  strains  in  general,  is  indicated  by  the 


BACJLLUS-COLI-LlKE  BACTERIA   FROM   FECES  AND   SEWAGE  23 

I 


FLcrrin 
H  By  Cou-Lncc  BACTEKIA  FKOM  /In/HAL  Feces 

similar  results  obtained  with  the  11  cultures  from  the  collection  of  the 
American  Museum  of  Natural  History.  Four  of  the  museum  strains 
were  positive  and  7  negative  for  the  Voges-Proskauer  reaction. 

The  average  quantities  of  acid  formed  from  different  substances 
by  the  Voges-Proskauer-positive  and  -negative  strains  obtained  from 
the  museum  collection  and  isolated  in  this  laboratory  are  shown  in 
Table  11  and  Plot  IV. 

The  museum  strains,  both  Voges-Proskauer-positive  and  -negative, 
form  less  acid  from  lactose  than  the  organisms  freshly  isolated  from 
animals  and  sewage,  but  in  all  other  substances  tested  the  differences 
between  the  museum  and  freshly  isolated  strains  are  inappreciable. 
To  infer  that  the  museum  strains  have  lost  their  power  to  ferment  lac- 
tose does  not  offer  an  adequate  explanation  of  all  the  phenomena,  for 
it  becomes  necessary  to  explain  why  the  organisms  should  single  out 
and  taboo  lactose  while  retaining  their  power  to  form  acid  from  the 
simpler  and  more  easily  attacked  monosaccharids.  as  well  as  the  more 


24 


MAX    LEVINE 


difficultly  fermented  disaccharids,  trisaccharid,  alcohols,  and  glucosid 
studied.  No  attempt  will  therefore  be  made  to  explain  this  phenome- 
non with  lactose,  except  to  suggest  that  it  may  possibly  be  attributed 
to  the  small  number  of  museum  strains  studied. 

An  inspection  of  Plot  IV  and  Table  11  indicates  that  all  the  167 
strains  studied  considered,  the  Voges-Proskauer-positive  organisms 
form  less  acid  from  glucose  than  do  the  Voges-Proskauer-negative 
strains,  and  about  equal  quantities  from  galactose,  mannitol,  and  lac- 
tose. In  all  other  test  substances — maltose,  salicin,  raffinose,  dulcitol, 
glycerol,  and  sucrose — the  Voges-Proskauer-positive  strains  give  rise 
to  more  acid,  the  excess  increasing  in  the  order  named.  The  differ- 

TABLE    11 

ACID-PRODUCTION   IN  FERMENTABLE  SUBSTANCES  BY    VOGES-PROSKAUER-POSITIVE   AND    -NEGATIVE 

BACILLUS-COLI-LlKE    BACTERIA 


Percentage  of  Normal  Acid 

Excess  of  Acid 
(in  Percentage  of 
Normal)  by  the  V.  P.+ 
Organisms 

American-Museum 
Test  Substance                      Strains 

Levjne's 
Strains 

V.  P  .  — 

V.  P.  + 

V.  P.  — 

V.  P.  + 

American- 
Museum 
Strains 

Levine's 
Strains 

Glucose                                      1  82 

1.52 
1.28 
0.85 
1.41 
1.01 
1.52 
1.22 
1.27 
1.15 
1.38 

1.82 
1.36 
1.31 
1.31 
0.75 
0.74 
0.96 
0.70 
0.81 
0.94 

1.46 
1.21 
1.26 
1.48 
1.12       •' 
1.57 
1.32 
1.28 
1.30 
1.28 

—  .30 
—  .03 
—  .11 
+  .04 
'••     +  .35 
+  .81' 
+  .43 
+  .69 
+  .32 
+  .38 

—  .36 
—  .15 
—  .05 
+  .17 
+  .37 
+  .83 
+  .36 
+  .58 
+  .49 
+  .34 

Galactose  1.31 

Lactose                                     0  96 

Mannitol         137 

Maltose  0.66 

Sucrose                                      0  71 

Raffinose  0.79 

Glycerol                                     0  58 

Dulcitol   0.83 

Salicin                                       1  00 

ences  obtained  in  salicin,  raffinose,  and  possibly  glucose,  are  probably 
not  significant,  on  account  of  the  variations  observed  in  acid-produc- 
tion from  these  substances. 


THE  SUPPOSED  Loss  OF  FERMENTING  POWER  BY  B.  COLI  IN  ITS 
PASSAGE  THROUGH  SEWAGE 

Browne  observed  that  colon-bacillus-like  organisms  from  oysters 
formed  less  acid  from  glucose  than  did  similar  organisms  derived 
from  man.  He  concludes :  "The  bacillus  coli  isolated  from  f eces,  both 
from  laboratory  assistants  and  from  the  immigrants  of  the  5.  S.  Roma, 
produced  more  acid  in  dextrose  and  lactose  broth  than  the  colon  bacil- 
lus isolated  from  oysters.  This  seems  to  indicate  that  Bacillus  coli 
loses  some  of  its  ability  to  ferment  carbohydrate  with  the  production 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  25 

of  acid  during  the  journey  from  the  intestinal  tract  to  the  oysters." 
He  states,  however,  that  in  his  laboratory  experiments  he  was  unable 
to  cause  a  reduction  in  fermenting  power  even  after  long  periods  (8 
weeks)  of  storage  in  sea  water. 

It  appears  from  this  study  that  a  very  plausible  explanation  of 
Browne's  results  is  that  Voges-Proskauer-positive  organisms  were 
among  his  oyster  strains.  Such  organisms  are  very  rare  in  feces, 
but  not  uncommon  in  sewage  and  soil  washings.  The  admixture  of, a 


ftc/d-Ffroe/uct/on  by  Voges  - 

i  Co//'- Li  fee  Bacter/a 

few  Voges-Proskauer-positive  organisms  in  a  collection  of  colon- 
bacillus-like  strains  would  decrease  the  mean  amount  of  acid  formed 
from  glucose  and  raise  the  titer  of  that  from  sucrose.  The  oyster 
strains  employed  by  Browne  formed  less  acid  from  glucose,  and  some- 
what more  from  sucrose,  than  the  fecal  strains,  thus  confirming  to 
some  extent  the  inference  that  the  differences  he  observed  were  due 
to  an  admixture  of  a  few  Voges-Proskauer-positive  organisms  rather 
than  to  a  loss  of  fermenting  power  by  colon-bacillus-like  strains  in 
their  passage  through  sewage. 


26  MAX    LEVINE 

THE  SUBSTITUTION  OF  QUANTITATIVE  ACID-PRODUCTION  FOR  GAS- 
FORMATION  AS  A  DIFFERENTIAL  INDEX  IN  STUDIES 

ON   B.    COLI 

Kligler6  suggests  that  quantitative  acid-production  be  substituted 
for  gas-formation  as  an  index  of  fermentation.  He  points  out  that 
in  standard  meat-infusion  sugar-freed  carbohydrate  broth  media  there 
is  a  rather  sharp  dividing  line  between  acid-producers  and  nonacid- 
producers  at  1.5%  normal  acid,  and  that  quantitative  gas-production 
is  variable  and  unreliable.  Of  course  it  is  agreed  that,  as  a  quantitative 
test,  gas-formation  as  ordinarily  determined  in  the  Smith  or  Durham 
tube  is  of  little  value;  as  a  qualitative  test,  however,  it  may  be  of 
considerable  significance.  If  a  culture  is  inoculated  into  sugar  broth 
and  gas  is  formed,  while  no  gas  is  produced  in  plain  broth,  the  organ- 
ism would  most  certainly  be  regarded  as  a  fermenter  irrespective  of 
whether  more  or  less  than  1.5%  acid  is  formed. 

Kligler  apparently  regards  such  an  organism  as  a  nonfermenter, 
for  he  says :  "The  members  of  the  proteus  group,  on  the  other  hand, 
produced  from  10  to  20  per  cent,  gas  in  lactose  broth  tho  at  no  time 
did  they  produce  more  than  1.0  percent  normal  acid,"  and  he  later 
records  this  group  as  lactose-negative.  It  is  not  the  ^intention  to 
debate  at  this  point  whether  B.  proteus  is  a  lactose- fermenter  or  not, 
but  it  should  be  pointed  out  that  to  say  that  an  organism  which  forms 
gas  from  a  carbohydrate  is  a  nonfermenter  because  the  acid  titer  is 
low,  introduces  confusion  into  the  already  much  maligned  and  abused 
term  "fermentation."  The  low  titer  might  be  due  to  a  secondary, 
alkali-production  which  masks  the  acid,  as  suggested  by  Rogers.  It 
has  been  repeatedly  observed  in  this  laboratory  that  B.  aerogenes  in 
peptone  dipotassium-phosphate  solution  containing  1  or  2%  glucose, 
may  be  acid  to  methyl  red  after  24  hours'  incubation,  but  alkaline 
after  from  48  to  96  hours  at  37  C. 

Rogers,  Clark,  and  Evans7  also  determined  titratable  acid  and 
selected  1%  normal  acid  as  the  point  of  demarcation  between  fer- 
menters  and  nonf  ermenters,,  but  they  point'  out  the  possible  errors  in 
acid-determination  and  give  precedence  to  gas-formation  as  indicated 
in  the  following: 

"Under  certain  circumstances  which  have  not  yet  been  definitely  deter- 
mined, the  acid  from  the  fermentation  of  sugar  may  be  masked  by  a  secondary 
alkaline  production,  sufficient  in  some  cases  entirely  to  obscure  the  acid  for- 

6  Jour.  Infect.  Dis.,  1914,  15,  p.  137. 

7  (a)  Jour.  Infect.  Dis.,  1914,  14,  p.  411;  (b)  IS,  p.  100;  (c)  1915,  17,  p.  137. 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  27 

mation.  In  one  small  group  of  this  collection,  the  lactose  broth  tubes  at  the 
end  of  seven  days  were  only  slightly  more  acid  than  the  blank,  altho  all  of 
the  cultures  gave  gas  in  lactose  bile.  In  no  case  was  the  titration  of  the  cul- 
ture less  than  that  of  the  blank,  altho  this  was  usually  the  case  with  broths 
in  which  there  was  no  fermentation.  Where  positive  evidence  of  the  fer- 
mentation of  a  sugar  was  obtained  in  another  way,  the  negative  evidence  of 
the  titration  was  disregarded  and  in  the  correlation  tables  the  culture  was 
included  with  the  positive  reactions.  If,  for  instance,  the  titration  of  lactose 
broth  was  negative,  while  the  lactose  bile  fermentation  tubes  showed  gas,  the 
cultures  were  considered  to  be  lactose  positive." 

Acid-production  should  not  be  given  precedence  over  gas-forma- 
tion. They  may  be  independent  characters.  If  however,  after  care- 
ful studies,  it  appears  that  there  is  a  marked  correlation  between  quan- 
titative acid-production  and  qualitative  gas-formation,  then  it  may  be 
feasible  to  supplement,  if  not  substitute,  the  gas  test  by  the  acid  test. 
In  that  event,  the  line  of  demarcation  between  fermenters  and  non- 
fermenters  would  have  to  be  determined  for  the  medium  employed. 
In  this  study,  with  peptone  water  containing  1%  carbohydrate,  non- 
fermenters  rarely  produced  as  much  as  0.2%  normal  acid. 

Another  point  of  disagreement  as  to  acid-production  by  B.  coli  is 
the  maximal  amount  of  acid  formed.  Kligler,6  using  meat-infusion 
media,  often  obtained  titers  of  4%  normal  acid  or  more,  and  similar 
results  have  been  recorded  by  Rogers.7  Browne,1  however,  using 
Liebig's  meat-extract  media,  states  that  the  limiting  acidity  for  B.  coli 
is  2.4%  normal  acid  as  determined  by  titration  with  phenolphthalein. 
Winslow  and  Walker8  determined  the  acid-production  in  12  substances 
by  B.  coli.  The  maximal  acidity  observed  was  0.45  c.c.  N/20  NaOH 
to  the  cubic  centimeter  of  culture  medium,  or  2.25%  normal  acid. 

In  the  study  recorded  here,  with  peptone  water  as  the  basic  medium, 
the  results  are  in  entire  accord  with  Winslow  and  Walker's,  and  with 
Browne's.  Of  more  than  2500  titrations,  none  showed  more  than 
2.4%  normal  acid. 

The  difference  in  acid-production  observed  by  various  investigators 
is  probably  due  to  differences  in  the  composition  of  the  media 
employed.  It  is  now  well  established  that  more  acid  is  formed  in  meat- 
infusion  broth  than  in  beef-extract  broth.  In  media  containing  much 
phosphates,  as  yeast  water,  even  more  acid  is  formed  than  in  meat- 
infusion  broth.  Within  certain  limits  the  amount  of  acid  formed,  as 
determined  by  titration  with  phenolphthalein,  is  a  function  of  the 
amount  of  buffer  substances  (as  K2HPO4  amino-acids,  extractives, 
etc.)  present  in  the  culture  medium.  Acid  is  formed  until  a  certain 

8  Science,    1907,   26,   p.    797. 


28  MAX    LEVINE 

H-f— ion  concentration  is  reached.  The  ratio  of  the  total  titratable 
acid  formed  to  the  maximal  or  limiting  H-f-ion  concentration  is  not 
constant,  but  varies,  within  limits,  with  the  amount  of  buffer  materials 
present  in  the  medium. 

The  limiting  H-| — ion  concentration  may  be  an  index  of  (1)  the 
resistance  of  an  organism  to  acid  (H-f-  ions),  or  (2)  the  point  of 
equilibrium  between  the  decomposing  carbohydrate  and  its  end  prod- 
ucts under  the  influence  of  an  organism. 

If  the  limiting  H-| — ion  concentration  in  glucose  broth  is  such  as 
to  inhibit  further  growth  of  the  organism,  then  the  organism  will  die 
and  the  H-| — ion  concentration  will  rejpiain  constant.  This  seems  to 
be  the  course  of  events  with  the  Voges-Proskauer-negative  group. 
With  the  Voges-Proskauer-positive  organisms  the  H-] — ion  concentra- 
tion rises  to  a  maximum  and  then  decreases,  tjhe  medium  becoming 
alkaline  to  methyl  red.  Under  these  conditions  it  is  inferred  that  the 
maximal  H-f^ion  concentration  is  a  measure  of  the  point  of  equili- 
brium between  glucose  and  its  end  products  under  the  influence  of  the 
organism  in  question. 

It  may  be  further  considered  that  after  the  limiting  H+- ion  con- 
centration is  reached,  the  organism,  if  not  destroyed,  will,  if  capable, 
attack  the  peptones  forming  alkali.  Some  of  the  free  acid  becomes 
neutralized  and  more  carbohydrate  may  be  decomposed.  The  H-J-- ion 
concentration  would  remain  constant  as  long  as  there  is  any  ferment- 
able carbohydrate  present.  If  this  assumption  is  correct,  then  an 
increase  of  the  carbohydrate  should  retard  the  reversion  from  an  acid 
to  an  alkaline  reaction.  This  is  exactly  what  takes  place.  In  some 
work  now  in  progress  it  has  been  found  that  Voges-Proskauer-positive 
strains  were  alkaline  to  methyl  red  after  24  hours'  incubation  in  0.5% 
peptone  dipotassium-phosphate  solution  containing  0.5%  glucose.  In 
the -same  medium  with  1%  glucose,  the  reaction  was  acid  after  24 
hours  but  alkaline  after  from  48  to  72  hours.  With  2%  glucose,  the 
acid  reaction  persisted  until  the  4th  or  5th  day.  With  5%  glucose 
there  was  no  reversion  to  an  alkaline  reaction  even  after  several  weeks. 

THE   CORRELATION   OF  ACID-  AND  GAS-FORMATION 

Table  12  shows  the  relation  of  gas-production  to  the  amount  of 
acid  formed  from  sucrose,  raffinose,  dulcitol,  glycerol,  and  salicin. 
The  other  test  substances  are  not  indicated  because  they  were  invari- 
ably fermented  with  production  of  gas.  Cultures  are  regarded  as  gas- 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  29 

formers  if  gas  is  observed  in  the  closed  arm  irrespective  of  the 
quantity. 

Table  12  indicates  that  with  sucrose,  raffinose,  and  dulcitol,  acid- 
and  gas-production  after  36  hours'  incubation  at  37  C.  are  strikingly 
correlated.  Of  80  organisms  which  fail  to  form  gas  from  sucrose,  79 
(98.8%)  form  less  than  0.2%  normal  acid,  and  the  remaining  culture 
forms  only  0.2%  normal  acid.  Among  the  75  organisms  which  do 
give  gas  from  sucrose,  8  (10.6%)  form  more  than  0.4  but  less  than 
0.6%,  48  (64%)  give  between  0.6  and  0.79%,  and  the  remaining  19 
strains  (25.4%)  form  more  than  0.8%  normal  acid.  There  is  no  over- 
lapping whatever  between  the  amounts  of  acid  produced  by  the  gas- 
formers  and  the  nongas-formers.  To  summarize:  of  the  80  strains 
which  fail  to  produce  gas,  none  form  more  than  0.2%  normal  acid, 
while  among  the  74  gas-producers  the  minimal  amount  of  acid  pro- 
duced in  peptone  solution  containing  1%  sucrose  is  more  than  0.4% 
normal  acid. 

A  similar  correlation  is  observed  between  acid-  and  gas-formation 
in  1%  dulcitol  in  peptone  solution.  Of  88  strains  that  do  not  form 
gas,  86  (97.8%)  give  less  than  0.2%  normal  acid.  The  remaining  two 
organisms  form  0.3%  and  0.4%  normal  acid.  Among  the  67  gas- 
formers,  however,  there  are  only  2  (3%)  that  produce  less  than  0.4% 
normal  acid. 

The  correlation  of  acid-  and  gas-production  in  peptone  raffinose 
solution  is  also  very  marked ;  79  produce  gas  and  77  fail  to  form  gas 
from  raffinose.  Of  the  nongas-formers  72  (93.5%)  form  less  than 
0.2%  normal  acid;  3  organisms  (3.9%)  between  0.2%  and  0.6%  acid; 
and  2  cultures  (2.6%)  more  than  0.8%  acid.  Among  the  gas  formers 
1  culture  (1.3%)  produces  no  acid,  while  2  others  (2.5%)  form  less 
than  0.6%  normal  acid.  The  other  76  gas-formers  (96.2%)  form 
more  than  0.6%  normal  acid. 

With  glycerol  and  salicin  the  correlation  of  acid-production  and 
gas-formation  is  not  nearly  so  striking  as  it  is  with  sucrose,  dulcitol, 
or  raffinose. 

Gas  is  formed  from  glycerol  by  118  of  the  cultures  after  72  hours' 
incubation,  while  38  organisms  do  not  form  gas.  Of  the  gas-formers, 
16  (13.6%)  produce  0.4-0.59%  normal  acid  as  compared  with  23 
(60.6%)  of  the  nongas-formers,  while  61  (51.7%)  of  the  former  and 
5  (13.2%)  of  the  latter  give  0.6-0.79%  normal  acid.  One  organism 
which  does  not  form  gas  yields  more  than  0.8%  normal  acid. 


30 


MAX    LEVINE 


TABLE     12 
RELATIONSHIP  BETWEEN   QUANTITATIVE  ACID-PRODUCTION  AND  GAS-FORMATION  BY  B.  COLI 


Test  Substance 

Gas 

Percentage  or  Normal  Acid 

0-0.19 

0.20-0.39 

0.40-0.59 

0.60-0.79 

0.80  or 
more 

Sucrose  

+ 

J  No. 

1    % 

f  No. 

1    % 

0 

79 
98.8 

0 

1 

1.2 

8 
10.6 

0 

48 
64.0 

0 

19 
25.4 

0 

Raffinose  

: 

'  + 

f  No. 
1    % 

f  No. 

1    % 

1 
1.3 

72 
93.5 

0 

1 
1.3 

2 
2.5 

2 
2.6 

18 
22,8 

0 

58 
73.4 

9. 

2.6 

Dulcitol 

+ 

f  No. 
1    % 

f  No. 

1    % 

0 

86 
97.8 

2 
3.0 

1 
1.1 

5 
7.5 

1 
1.1 

23 
34.3 

0 

37 
55.2 

0 

r    i_ 

I  No. 
i 

0 

0 

1 

19 

82 

Salicin  

1  % 

[  No. 

43 

1 

10 
3 

18.6 
6 

80.4 
1 

1  % 

79.7 

1.8 

5.6 

11.1 

1.8 

Glycerol 

+ 

r  NO. 
i  % 

r  NO. 
1  % 

0 

4 
10.5 

0 

5 

13.2 

16 
13.6 

23 
60.5 

61 
51.7 

5 
13.2 

41 
34.7 

1 

2.6 

BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  31 

Salicin  is  fermented,  with  gas-formation,  by  102  organisms  after 
72  hours  at  37  C,  while  54  strains  do  not  form  gas.  Among  the  non- 
gas-formers,  10  (18.5%)  produce  0.4-0.8%  normal  acid,  whereas 
this  quantity  of  acid  is  also  formed  by  20  (19.6%)  of  the  gas-formers. 

It  appears  from  Table  12  that  under  the  conditions  of  these  experi- 
ments, acid-production  in  sucrose,  dulcitol,  and  raffinose  is  well  corre- 
lated with  the  presence  or  absence  of  gas.  With  salicin  the  correlation 
is  not  so  marked,  while  with  glycerol  the  line  of  demarcation  between 
gas-formers  and  nongas-formers,  as  indicated  by  the  quantity  of  acid 
produced,  is  very  indistinct.  The  substitution  of  quantitative  acid- 
production  for  gas-formation  would  therefore  be  particularly  undesir- 
able when  working  with  glycerol. 

These  results  are  well  in  accord  with  those  of  Winslow  and 
Walker,8  who  observe:  "Gas-formation  coincided  with  acidity  except 
in  the  case  of  dextrin."  Unfortunately,  acid-formation  in  dextrin  was 
not  determined  in  this  study,  and  Winslow  and  Walker  did  not  employ 
salicin  or  glycerol. 

CHARACTERISTICS   OF   ORGANISMS    FROM    THE   DIFFERENT    SOURCES 

When  this  study  was  begun  (1915),  motility  and  fermentation  of 
dextrin  and  starch  were  regarded  as  of  little  significance  and  hence 
these  tests  were  omitted.  In  the  following  year  (1916)  the  possible 
significance  of  the  reactions  was  realized  and,  as  the  cultures  were 
still  available,  they  were  tested  out.  Motility  was  determined  in  a 
soft  agar  medium  consisting  of  nutrient  broth  and  0.5%  agar. 

In  Table  13  are  shown  the  number  and  percentage  of  organisms 
giving  positive  reactions  with  the  various  tests.  Glucose,  galactose, 
mannitol,  maltose,  and  lactose  are  fermented  by  all  strains,  with  gas- 
production.  Inulin  is  not  fermented  by  any  of  the  organisms,  and 
gelatin  is  uniformly  negative  in  20  days  at  20  C.  Gas  is  formed  from 
glycerol  by  76.2%,  from  salicin  by  66.1%,  from  raffinose  by  50.7%, 
from  sucrose  by  48.7%,  from  dulcitol  by  43.6%,  from  dextrin  by  5.1%, 
and  from  starch  by  4.5%.  The  Voges-Proskauer  reaction  is  given  by 
5.8%,  indol  is  produced  by  91.1%,  and  61.5%  are  motile. 

Table  14  shows  the  characters  of  organisms  isolated  from  different 
sources.  Characters  which  are  negative  or  positive  for  all  strains  are 
omitted. 

Several  things  are  evident.  Organisms  giving  a  positive  Voges- 
Proskauer  reaction  or  gas  from  dextrin  and  starch  were  obtained 


32 


MAX    LEVIN E 


TABLE     13 

GAS-FORMATION  AND  OTHER  CHARACTERISTICS  OF  BACILLUS-COLI-LIKE  BACTERIA  FROM   VARIOUS 

ANIMALS   AND    SEWAGE 


Character 

Number 
Positive 

*  Percentage 
Positive 

Motility          

96 

61  5 

Gelatin 

0 

0 

Indol 

14-7 

91  1 

Voges-Proskauer  

9 

5.8 

Glucose 

156 

100 

Galactose.  ..        

156 

100 

Mannitol  

156 

100 

Dulcitol 

68 

43  6 

Glycerol 

118 

76  .^ 

Maltose  

156 

100 

Lactose 

156 

100 

Sucrose  .         ... 

48  7 

Raffinose  

79 

507 

Salicin 

102 

66  1 

Dextrin  .          i. 

8 

5  1 

Inulin 

o 

o 

Starch 

7 

4  5 

only  from  sewage.  This  must  not  be  taken  to  mean  that  such  organ- 
isms are  entirely  absent  from  the  other  sources,  but  it  certainly  indi- 
cates that  they  are  extremely  scarce  in  feces  of  the  animals  studied. 

Salicin  is  fermented  by  95%  of  the  bovine  strains,  and  by  8  (89%) 
of  the  9  Voges-Proskauer-positive  strains  from  sewage.     Organisms 


TABLE     14 
MOTILITY  AND  OTHER  REACTIONS  OF  BACILLUS-COLI-LIKE  BACTERIA  FROM   DIFFERENT   SOURCES 


Source 

Horse 

Sheep 

Cow 

Pig 

Man 

Sewage 

V.  P.  — 

V.  P.  4- 

Number  of  strains  

19 

100.0 
0.0 
100.0 
79.0 
73.8 
68.5 
84.3 
73.8 
0.0 
0.0 

22 
Pe 

77.3 
0.0 
100.0 
95.5 
100.0 
50.0 
62.0 
68.3 
0.0 
0.0 

20 
rcentage 

80.0 
0.0 
100.0 
50.0 
50.0 
50.0 
95.0 
95.0 
0.0 
0.0 

31 
of   Positi 

93.7 
0.0 
93.7 
32.3 
32.3 
42.0 
74.2 
58.1 
0.0 
0.0 

25 
ve  Reacti 

32.0 
0.0 
84.0 
12.0 
16.0 
20.0 
64.0 
44.0 
0.0 
0.0 

SO 
ons 

20.0 
0.0 
83.5 
26.6 
33.3 
433 
70.0 
60.0 
0.0 
0.0 

9 

11.1 
100.0 

e;6.7 

.  100.0 
100.0 
33.3 
88.9 
•£8.9 
88.9 
77.8 

Motility 

Voges-Proskauer  reaction  
Indol  

Sucrose 

Raffinose  .         

Dulcitol 

Glycerol 

Salicin 

Dextrin     * 

Starch      

from  other  sources  attack  salicin  less  readily — horse  73.8%,  sheep 
68.3%,  pig  58.1%,  man  44%,  and  sewage  (Voges-Proskauer-negative 
strains)  56.7%.  This  glucosid  was  used  by  MacConkey,3  who  did  not 
regard  its  employment  worth  while  for  classification  purposes. 
Recently  (1914)  Kligler6  suggested  that  salicin  displace  dulcitol  in 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  33 

subdivision  of  the  colon-bacillus  group,  but  Rogers7  questions  the 
value  of  salicin  in  view  of  the  very  large  number  of  his  strains  which 
attacked  it.  In  this  connection  it  might  be  well  to  point  out  that 
organisms  studied  by  Rogers  consisted  of  bovine  strains,  grain  strains 
(probably  Voges-Proskauer-positive  organisms),  and  milk  strains 
(which  may  be  considered  for  the  most  part  as  a  mixture  of  bovine 
and  grain  strains).  In  view  of  the  results  obtained  here  with  bovine 
and  Voges-Proskauer-positive  strains,  and  by  Kligler  with  Voges- 
Proskauer-positive  strains,  it  would  be  expected  that  more  than  90% 
of  Rogers'  cultures  would  attack  salicin.  It  appears  then  that  salicin- 
fermentation  is  somewhat  correlated  with  the  source. 

Glycerol  is  also  fermented  by  almost  all  the  Voges-Proskauer- 
positive  and  bovine  strains  and  less  frequently  by  organisms  from  the 
other  animals,  but  the  difference  is  less  marked  than  with  salicin. 

Dulcitol  is  only  occasionally  fermented  by  the  human  and  Voges- 
Proskauer-positive  strains,  but  there  seems  to  be  very  little  relation 
between  dulcitol-fermentation  and  the  animal  source. 

Indol-production  is  not  correlated  with  the  animal  source. 

In  motility  there  is  a  marked  contrast  between  the  strains  from 
horse,  sheep,  cow,  and  pig  on  the  one  hand,  and  those  from  man  and 
sewage  on  the  other.  Less  than  one-third  of  the  sewage  and  human 
strains  are  motile,  as.  compared  with  more  than  four-fifths  of  the 
other  animal  strains.  McWeeney9  found  nonmotile  B.  coli  abundant 
in  feces,  and  notes  that  Stocklin  also  had  observed  many  nonmotile 
forms  among  fecal  strains.  Just  what  significance  is  to  be  attached 
to  motility  is  hard  to  say  at  present,  because  so  few  bacteriologists 
determine  this  character  in  routine  work.  MacConkey,  however, 
strongly  advocates  the  test.  As  determined  in  the  0.5%  agar  medium 
the  motility  test  is  simple,  quick,  and  not  at  all  burdensome. 

Sucrose  and  raffinose  are  so  well  correlated  that  a  consideration 
of  either  will  suffice  for  both.  The  Voges-Proskauer-positive  and 
sheep  strains  are  practically  all  sucrose- fermenters  (100%  and  95.5% 
respectively).  Of  the  horse  strains  79%,  and  of  the  organisms  from 
the  cow  50%  form  gas  from  sucrose;  only  32.3%  of  strains  from  the 
pig,  26.6%  of  those  from  sewage  (Voges-Proskauer-negative  strains), 
and  12%  of  those  from  man  form  gas  from  sucrose.  That  such  a 
small  number  of  human  strains  attack  sucrose  is  particularly  interest- 
ing, and  a  review  of  the  literature  indicates  that  similar  results  have 

9  Cited  by  Prescott  and  Winslow,  Elements  oi:   Water  Bacteriology,   1913. 


34 


MAX    LEVINE 


TABLE     15 

FERMENTATION   OF   SUCROSE   BY   BACILLUS-COLI-LIKE  BACTERIA  FROM   HUMAN   FECES 


Investigators 

Number  of 
Organisms 
Studied 

Number  of 
Sucrose 
Fermenters 

Percentage  of 
Sucrose 
Fermenters 

Houston,11  1902-3  

100 

30 

30 

MacConkey,3  1905  and  1909  

419 

142 

33.9 

Ferreira,12  Horta,  Paredes,  1908  . 

117 

44 

37.6 

Winslow8  and  Walker,  1907  

25 

8 

32 

Howe,10  1912  

540 

324 

60 

Clemesha,13  1912  

1200 

348 

29 

Browne  -1  1915 

175 

20 

11  3 

Levine,4  1916               

25 

3 

12 

Total 

2601 

919 

353 

been  obtained  by  previous  investigators.  In  Table  15  is  shown  the  pro- 
portion of  sucrose-fermenters  obtained  from  human  feces  by  different 
investigators.  Howe10  found  60%  of  540  Bacillus-coli-like  organisms 
to  be  sucrose  fermenters,  but  the  other  investigators  usually  found 
twice  as  many  nonfermenters  as  fermenters.  Of  2601  cultures  of 
human  colon  bacilli  studied  by  various  observers,  at  different  times 
and  in  different  countries,  only  35.3%  fermented  sucrose. 

In  connection  with  the  study  reported  here  it  should  be  noted  that 
the  number  of  human  strains  isolated  is  small,  and  that  they  were  col- 
lected in  the  winter.  Clemesha,13  and  also  Browne,1  call  attention  to 
"epidemics"  of  certain  types  of  B.  coli,  and  to  seasonal  variations. 
These  phases  need  further  investigation. 

CONCLUSIONS 

In  studies  on  quantitative  acid-production  the  average  should  be 
supplemented  with  a  statement  of  its  deviation  measures ;  the  unquali- 
fied average  may  lead  to  a  misconception  of  the  acid-producing  prop- 
erties of  a  group  of  organisms. 

Quantitative  acid-production  in  glucose,  galactose,  maltose,  lactose, 
sucrose,  raffinose,  salicin,  inulin,  mannitol,  dulcitol,  and  glycerol,  is  not 
a  reliable  index  for  differentiating  colon-bacillus-like  bacteria  derived 
from  pig,  horse,  sheep,  cow,  or  man. 

The  MacConkey  types  are  indistinguishable  on  the  basis  of  quan- 
titative acid-production  in  the  fermentable  carbohydrates,  the  alcohols, 
and  the  glucosid  studied. 

*°  Science,   1912,  35,  p.  225. 

11  Suppl.  to  32nd  Ann.  Rep.  containing  Rep.  of  Med.  Officer  for  1902-1903,  p.   511. 

12  Arch.   d.   Real  Inst.   Bacteriol.  Camara  Pestana,   1908,   2,  p.    153. 
«  Jour.  Hyg.,  1912,  12,  p.  463. 


BACILLUS-COLI-LlKE  BACTERIA  FROM   FECES  AND   SEWAGE  35 

The  Voges-Proskauer-positive  strains  (aerogenes-cloacae  group) 
form  somewhat  less  acid  from  glucose,  but  more  acid  from  maltose, 
sucrose,  glycerol,  and  dulcitol,  and  possibly  also  from  raffinose  and 
salicin,  than  do  the  Voges-Proskauer-negative  strains  (colon-bacillus 


Acid-formation  should  not  be  given  precedence  over  gas-formation 
in  studies  on  B.  coli,  for  the  acid  may  be  masked  by  a  secondary  alkali- 
production.  In  general,  however,  acid-production  is  accompanied  by 
gas-formation.  With  sucrose,  dulcitol,  and  raffinose,  acid-production 
and  gas-formation  are  almost  perfectly  correlated.  The  correlation  is 
less  marked  in  the  case  of  salicin,  while  the  line  of  demarcation 
between  gas-formers  and  nongas-formers,  as  indicated  by  quantitative 
acid-production  from  glycerol,  is  very  indistinct. 

Practically  all  Voges-Proskauer-positive  and  bovine  strains  attack 
salicin  with  liberation  of  gas.  This  glucosid  is  fermented  less  fre- 
quently by  the  organisms  from  pig,  horse,  sheep,  man,  and  sewage 

Gas  is  formed  from  sucrose  as  follows:  Voges-Proskauer-positive 
(  Voges-Proskauer-negative  strains  )  . 

strains  100%,  sheep  95.6%,  horse  79%,  cow  50%,  pig  32.3%,  sewage 
(Voges-Proskauer-negative  strains)  26%,  and  human  strains  12%. 

Of  2601  human  strains  of  B.  coli  studied  by  different  investigators, 
in  various  countries  and  at  different  times,  only  35.3%  have  been 
sucrose-fermenters. 

Motility,  as  determined  in  semisolid  nutrient  agar,  seems  to  be  an 
important  character.  Only  32%  of  the  human  and  20%  of  the  Voges- 
Proskauer-negative  sewage  strains  are  motile,  as  compared  with 
93.7%  of  pig,  80%  of  cow,  77.3%  of  sheep,  and  100%  of  horse  strains. 


Reprinted  from  THE  JOURNAL  OF  BACTERIOLOGY 
•  VOL.  Ill,  No.  3,  May,  1918 


A  STATISTICAL  CLASSIFICATION  OF  THE  COLON- 
CLOACAE  GROUP 

MAX  LEVINE 

Iowa  State  College,  Ames,  Iowa 

Received  for  publication  May  17,  1917 

It  is  now  firmly  established  that  the  end  products  of  metab- 
olism, as  well  as  morphological  differences,  are  reliable  and  con- 
venient indices  for  differentiation  of  bacterial  species  and 
varieties.  In  the  group  of  coli-like  bacteria,  particular  attention 
has  been  paid  to  acid  and  gas  production  fron  various  fermentable 
substances. 

Theobald  Smith  (1893)  observed  that  some  B.  coli  ferment  su- 
crose and  therefore  recognized  two  forms. 

Durham  (1901)  suggested  the  name  B.  coli-communior  for  the 
sucrose  fermenting  variety  and  characterized  B.  lactis-aerogenes 
as  a  polysaccharid  fractor. 

MacConkey  (1905  and  1909),  whose  classification  has  been  most 
widely  employed,  subdivided  upon  gas  formation  from  sucrose 
and  then  from  dulcitol  thus  giving  4  main  types  generally  known 
as  the  B.  acidi-lactici  type  (sucrose  negative,  dulcitol  negative) ; 
the  B.  communis  type  (sucrose  negative  dulcitol  positive);  the 
B.  communior  type  (sucrose  positive,  dulcitol  positive),  and  the 
B.  aerogenes  type  (sucrose  positive,  dulcitol  negative).  Under 
each  type  are  recorded  a  number  of  varieties  according  to  gelatin 
liquefaction,  indol  production,  the  Voges-Proskauer  reaction, 
motility,  and  fermentation  of  inulin,  adonitol,  etc. 

Very  much  along  the  lines  of  the  MacConkey  classification  is 
that  of  Bergey  and  Deehan  (1908).  They  employed  8  characters 
— fermentation  of  sucrose,  dulcitol,  adonitol,  and  inulin;  gelatin 
liquefaction,  indol  production,  motility  and  Voges-Proskauer 
reaction — and  from  a  consideration  of  all  possible  combinations 

253 


254  MAX    LEVINE 

between  these  characters  recognized  the  possible  existence  of  256 
varieties  of  B.  coli. 

The  grouping  of  Jackson  (1911)  which  was  accepted  by  the 
American  Public  Health  Association  and  included  in  the  standard 
methods  for  1912,  is  very  similar  to  that  of  MacConkey,  but  here 
preference  is  given  to  dulcitol  over  sucrose  for  the  primary  divi- 
sion. Each  of  the  4  groups  thus  formed  is  subdivided  further 
on  raffinose  and  mannitol,  and  then  on  motility,  indol,  reduction 
of  nitrates,  and  gelatin  liquefaction. 

A  very  serious  objection  to  the  classifications  of  MacConkey, 
Bergey  and  Deehan,  and  Jackson,  is  their  extreme  flexibility. 
As  the  number  of  fermentable  substances,  or  other  characters 
observed,  increases,  the  number  of  " varieties"  increases  geomet- 
rically approaching  infinity.  The  number  of  "varieties"  is 
given  by  the  formula  2n  where  V  is  the  number  of  characters 
studied.  Thus  with  8  characters  there  are  256  possible  combi- 
nations; this  number  rises  to  1024  with  10  characters  and  to 
65,536  when  16  characters  are  observed.  The  absurdity  of 
regarding  each  character  as  of  similar  and  equal  differential 
value  is  thus  evident.  In  the  more  recent  studies  the  principle 
of  the  correlation  of  characters  has  been  emphasized. 

Howe  (1912),  from  a  statistical  study  of  630  strains  of  B.  coli 
isolated  from  human  feces,  concludes  that  dulcitol,  indol  pro- 
duction, nitrate  reduction,  etc.,  are  not  correlated  with  each  other 
nor  with  vigor  of  growth,  and  he  therefore  recognizes  only  the 
sucrose  positive  B.  communior  and  sucrose  negative  B.  communis. 

Rogers  and  his  associates,  (1914-1916)  studied  a  large  number 
of  coli-like  forms  from  milk,  grains,  and  bovine  feces,  and  con- 
clude that  two  distinct  groups  may  be  recognized  on  the  basis 
of  the  accurately  determined  gas  ratio — the  low  ratio  B.  com- 
munis-B*  communior  group  and  the  high  ratio  B.  aerogenes-B. 
acidi-lactici  group.  There  is  no  doubt  that  B.  communis  and  B. 
communior  are  low  ratio  strains  and  B.  aerogenes  of  the  high 
ratio  group  but  the  inclusion  of  B.  acidi-lactici  with  B.  aerogenes 
does  not  seem  justified,  and  I  believe  that  further  studies  will 
place  it  definitely  with  the  low  ratio  strains. 


CLASSIFICATION   OF   THE    COLON-CLOACAE    GROUP  255 

Kligler  (1915)  suggests  that  salicin  be  substituted  for  dulcitol, 
in  subdividing  coli-like  bacteria,  pointing  out  that  salicin  fermen- 
tation correlates  better  with  the  Voges-Proskauer  reaction  than 
does  dulcitol  decomposition.  He  thus  recognizes  a  sucrose  neg- 
ative, salicin  negative  group  (B.  acid-lactiti) ;  sucrose  negative, 
salicin  positive  group  (B.  communis) ;  sucrose  positive,  salicin 
negative  group  (B.  communior)  and  sucrose  positive,  salicin  posi- 
tive (B.  aerogenes) .  B.  cloacae  is  differentiated  from  B.  aerogenes 
by  its  inability  to  ferment  glycerol. 

The  characterization  of  B.  communior  as  salicin  negative  is 
probably  untenable.  The  term  B.  coli-communior  was  first  em- 
ployed by  Durham  to  describe  a  variety  of  B.  coli  which  fer- 
mented sucrose  and  which  was  motile.  Later  Ford  recognized  it 
as  a  species  B.  communior.  Such  organisms  usually  ferment 
salicin  as  will  be  shown  later  in  this  paper. 

Where  the  principle  of  correlation  has  been  employed  the  best 
correlated  character  has  apparently  been  picked  out  by  inspection 
of  the  data.  Inspection  is  a  tedious  and  difficult  procedure, 
entirely  inapplicable  where  the  number  of  characters  considered  is 
large,  and  it  does  not  permit  of  a  concise  statement  of  the  degree 
of  correlation  which  exists  between  different  reactions.  Consid- 
erable information  in  an  abstract,  concise,  and  workable, 
form  may  however  be  obtained  from  a  study  of  the  coefficients 
of  correlation. 

THE    COEFFICIENT   OF   CORRELATION 

Where  we  are  concerned  merely  with  the  presence  or  absence 
of  characters  the  coefficient  of  correlation  between  any  two  char- 
acters may  be  easily  determined.  Suppose  that  it  is  desired  to 
know  if  the  characters  X  and  Y  are  correlated  and  that  a  study 
of  a  number  of  organisms  showed  that  'a7  cultures  are  positive 
for  both  X  and  Y ;  '  b '  organisms  positive  for  X  but  negative  for 
Y,  V  cultures  are  negative  for  X  and  positive  for  Y;  and  'd' 
strains  are  negative  for  both  X  and  Y.  The  distribution  of  the 
organisms  is  first  tabulated  as  shown  below. 


256 


MAX    LEVINE 
Y 


X 


The  degree  of  association,  or  the  coefficient  of  correlation,  is 
then  expressed,  according  to  Yule,  by  the  formula 


ad  -  be 

ad  +  be 


(1) 


0 


If  '  ad '  is  equal  to  '  be '  the  coefficient  becomes     ,   .  ,     or  0 ; 

ad  -f  be 

which  indicates  that  there  is  no  correlation  whatever.     If  either 

ad 
'b'  or  'c'  is  zero  the  formula  becomes  — i  =  1;  indicating  a 

perfect  positive  correlation.     If  'a'  or  'd'  is  zero  then  we  have 

—be 

~r —  =  —  1 ;  showing  a  perfect  negative  correlation.     It  should  be 

observed  that  an  absolute  positive  correlation  exists  in  reality 
only  if  both  '  b '  and  '  c '  are  zero  and  an  absolute  negative  corre- 
lation when  both  V  and  '  d '  are  zero.  In  order  to  avoid  coeffi- 
cients of  1  or  —1  where  only  one  group — 'a',  'b,'  'c/  or  'd' — is 
zero.  Yule  gives  the  formula 


a 


+  d)-QH-c)  (a  +  b) 


(a  +  c) 


(2) 


In  practice,  however,  a  few  strains  are  almost  always  found  in 
each  of  the  four  groups  and  Yule  suggests  the  use  of  the  simpler 
formula  (1).  Some  caution  should  therefore  be  employed  in  in- 
terpreting coefficients  of  1  or  —1. 

For  this  study  it  was  assumed  that  if  the  coefficient  between 
two  characters  is  numerically  greater  than  0.5  they  may  be 
regarded  as  correlated,  but  if  less  than  0.3  there  is  probably  no 
association.  A  few  examples  of  correlation  coefficients  actually 
obtained  in  the  course  of  this  study  are  given  to  illustrate  the 
method  of  calculation. 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP 


257 


*   7 

43 

-  /3Z 

S 

7 


as 


+ 

— 

+    75 

22 

-  39 

46 

y- 

— 

+  B7 

/0 

-  76 

9 

&**r 


<C0rre/fr 


/a/  Corre/'sy 


The  principle  of  correlation  should  not  be  applied  indiscrimi- 
nately to  collections  of  data  for  systematic  purposes.  Certain 
characters  and  properties  have  been  universally  accepted  as 
reliable  and  appropriate  for  bacterial  differentiation;  thus,  stain- 
ing reactions  such  as  the  Gram  and  acid  fast  stains;  spore  forma- 
tion, aerobiosis  and  anaerobiosis,  hardly  need  to  be  bolstered 
up  by  correlation  with  other  characters  to  justify  their  taxo- 
nomic  value.  On  the  other  hand  the  significance  of  such  char- 
acters as  motility,  indol  production,  and  fermentation  of  certain 
substances,  is  still  debatable. 

Motility  is  regarded  by  many  as  a  highly  variable  property. 
Perhaps  it  is  in  reality  a  reliable  morphological  difference.  Cer- 
tainly if  it  could  be  shown  that  this  character  goes  hand  in  hand 
fwith  several  others,  more  reliance  and  attention  should  and 
would  be  given  to  motility.  The  same  is  true  of  the  indol  test. 
In  dealing  with  gas  formation  from  carbohydrates,  alcohols,  or 
poly  sac  charids,  the  question  naturally  arises  as  to  which  sub- 
stance should  be  given  preference  for  subdivision,  or  whether 
all  are  to  be  considered  of  equal  taxonomic  value.  The  lack  of  a 
criterion  for  determining  the  most  significant  fermentable  sub- 
stances has  led  to  considerable  confusion.  It  has  already  been 
pointed  out  how  subdivision  on  every  character  studied  results 
in  an  infinite  number  of  varieties.  Where  we  are  dealing  with  a 
number  of  characters  each  of  which  is  assumed  to  be  of  equal 
taxonomic  significance,  it  would  certainly  be  desirable  and 
advantageous  to  subdivide  on  that  character  which  gives  the 
greatest  amount  of  information  as  to  the  manner  in  which  the 
resulting  subgroups  react  with  respect  to  other  characters.  It 
is  under  such  circumstances  that  the  principle  of  correlation  of 
characters  may  be  legitimately,  conveniently,  and  advantage- 


THE  JOURNAL  OF  BACTERIOLOGY,  VOL.  Ill,  NO.  3 


258  MAX   LEVINE 

ously  employed.  It  may  be  recalled  that  the  differentiation  of 
the  colon-intermediate-typhoid  group  on  glucose  and  lactose 
fermentation  is  strikingly  correlated  with  pathogenicity. 

STATISTICAL   STUDY 

In  the  following  pages  is  evolved  a  classification  of  coli-like 
bacteria  based  primarily  upon  correlated  characters.  The  study 
is  made  upon  333  organisms  obtained  from  soil,  sewage  and  the 
feces  of  man,  horse,  sheep,  pig  and  cow. 

The  characters  considered  are  the  methyl-red  and  Voges- 
Proskauer  reactions,  indol  production,  motility,  gelatin  liquefac- 
tion and  gas  formation  from  sucrose,  raffinose,  dulcitol,  glycerol, 
salicin,  dextrin,  inulin  and  corn  starch.  Other  fermentable 
substances — lactose,  maltose,  galactose  and  mannitol — were  also 
observed  but  as  these  substances  were  all  attacked  with  gas  for- 
mation they  need  not  be  considered. 

The  investigations  of  Theobald  Smith,  Hardin,  Rogers  and 
others  indicate  distinctly  that  the  Voges-Proskauer  positive  or 
methyl-red  negative  strains  are  so  different  from  the  Voges- 
Proskauer  negative,  or  methyl-red  positive  organisms  with  respect 
to  the  end  products  of  carbohydrate  fermentation  that  subdivi- 
sion upon  these  characters  seems  justified.  Two  groups  are 
therefore  recognized,  the  methyl-red  positive,  Voges-Proskauer 
negative  or  B.  coli  group  and  the  methyl-red  negative,  Voges- 
Proskauer  positive  or  B.  aerogenes-B.  cloacae  group. 

METHOD    OF   STUDY 

The  organisms  in  each  of  the  two  groups  are  first  tabulated  as 
in  table  1  in  order  to  facilitate  the  calculation  of  the  correlation 
coefficients  which  are  then  determined  for  each  pair  of  characters 
and  recorded  as  indicated  in  table  IA.  In  choosing  between  any 
two  characters,  that  one  which  gives  the  highest  coefficient  of 
correlation  with  the  greatest  number  of  other  characters  is  selected 
for  subdivision.  For  the  resulting  subgroups  new  correlation 
tables  are  prepared  and  subdivision  again  made  as  above.  A 
point  is  very  quickly  reached  where  further  subdivision  upon 


CLASSIFICATION    OF    THE    COLON-CLOACAE    GROUP 


259 


correlated  characters  is  no  longer  feasible.  These  groups  are 
regarded  as  species  and  to  each  is  assigned,  as  far  as  possible,  the 
name  of  the  MacConkey  variety  which  it  most  resembles. 

TABLE  1 

Showing  correlation  of  characters  among  151  strains  of  the  Aerogenes-cloacae  group 


Si, 

'fffia 

rto. 

ll/fy 

fnc 

fo/ 

5ut 

rox 

faff, 

'vox 

fat 

vfol 

6fa 

ert 

5a/ 

'c/n 

/fcr 

fta 

f/7M 

to* 

5ra 

rt 

1 

/- 

83 

81 

2 

/3 

70 

S3 

79 

4 

// 

72 

7 

76 

62 

/ 

27 

56 

* 

79 

5 

78 

68 

& 

60 

33 

3$ 

65 

3 

66 

2 

34 

34 

62 

6 

67 

/ 

63 

5 

18 

41 

60 

6 

% 

+ 

8/ 

8 

89 

/5 

74 

86 

3 

83 

6 

14 

75 

8 

8/ 

87 

\ 

28 

6/ 

4 

84 

4 

85 

2 

60 

62 

3/ 

3/ 

62 

62 

3/ 

3/ 

61 

/ 

62 

62 

18 

36 

6/ 

/ 

i 

+- 

/3 

33 

/5 

3/ 

46 

46 

46 

24 

tf 

37 

9 

46 

39 

7 

'9 

3/ 

33 

/J. 

70 

35 

74 

3/ 

M 

<<£ 

3 

99 

6 

21 

84 

32 

73 

{03 

2 

f/ 

54 

/3 

89 

32 

73 

1 

+- 

83 

65 

86 

62 

46 

/O? 

<4S> 

/44 

4 

44 

{04 

69 

79 

#6 

2 

90 

58 

22 

J/7 

65 

83 

3 

3 

3 

3 

/ 

2 

/ 

2 

3 

3 

3 

3 

3 

^ 

•t- 

79 

66 

83 

62 

46 

99 

J44 

/ 

/45 

44 

/O/ 

68 

77 

/43 

2 

58 

27 

2/- 

J/5 

65 

9>- 

4 

2 

6 

6 

4 

2 

6 

/ 

5 

/ 

5 

6 

2 

4 

/ 

5 

6. 

1 

-f- 

// 

34 

/4 

3/ 

24 

2f 

44 

f 

44 

/ 

45 

3d 

7 

45 

37 

5 

// 

2t 

33 

/£ 

- 

7?. 

34 

75 

31 

22 

34 

J04 

2 

/Of 

£ 

i06 

3/ 

75 

/04 

2 

53 

53 

// 

89 

3£ 

74 

1 

+ 

7 

62 

8 

6/ 

37 

32 

69 

68 

/ 

38 

3/ 

69 

69 

66 

3 

20 

4/ 

63 

6 

76 

6 

8/ 

/ 

9 

73 

79 

3 

77 

5 

7 

7£ 

82 

80 

2 

24 

58 

Z 

79 

2 

8& 

I 

f 

82 

67 

Q7 

62 

46 

103 

/46 

3 

/£ 

6 

45 

/04 

60 

80 

149 

89 

60 

22 

1/9 

65 

84 

- 

/ 

/ 

2 

2 

2 

2 

2 

2 

£ 

/ 

/ 

/ 

£ 

i 

-f- 

27 

63 

28 

62 

39 

5/ 

90 

85 

2 

37 

53 

66 

£4 

89 

/ 

90 

2Z 

60 

65 

25 

56 

5 

6/ 

7 

54 

58 

3 

57 

£ 

8 

53 

3 

58 

60 

/ 

6/ 

60 

6/. 

2 

-t 

4 

/d 

4 

/6 

9 

/3 

22 

21 

/ 

10 

// 

20 

2 

2Z 

22 

2Z 

2/ 

/ 

- 

79 

4/ 

84 

36 

3/ 

d9 

//7 

3 

//5 

5 

& 

89 

41 

79 

//9 

/ 

60 

60 

tfft 

36 

84 

I 

+ 

5 

60 

4 

6/ 

33 

32 

65 

65 

33 

32 

63 

2 

65 

65 

?/ 

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78 

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d5 

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7J 

83 

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80 

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74 

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84 

£ 

25 

61 

/ 

04 

86 

*  Nine  strains  not  tested  in  inulin. 

THE    AEROGENES-CLOACAE    GROUP 

In  the  B.  aerogenes-B.  cloacae  group  are  included  all  strains 
which  gave  the  Voges-Proskauer  reaction — (practically  always 
alkaline  to  methyl-red)  and  10  cultures  which  fermented  starch 
with  gas  formation  but  did  not  react  typically  for  the  Voges- 
Proskauer  nor  methyl-red  tests.  There  are  151  organisms  in  the 
group,  9  of  which  were  obtained  from  sewage  and  the  rest,  142, 
from  soil. 


260 


MAX    LEVINE 


The  distributions  of  the  strains  with  respect  to  gelatin  lique- 
faction, motility,  indol  and  gas  formation  from  sucrose,  raffinose, 
dulcitol,  glycerol,  salicin,  dextrin,  inulin  and  starch  are  shown  in 
table  1.  Mannitol,  maltose,  lactose  and  galactose  were  always 
fermented  and  are  therefore  not  included. 

Gelatin  was  liquefied  by  83  (55  per  cent)  (observed  for  thirty- 
four  days  at  20°C.);  89  (59  per  cent)  were  motile;  46  (30.5  per 

TABLE  IA 
Coefficients  of  correlation  for  each  pair  of  characters  in  table  1 


Geibf//? 

f70fi//ry 

Mo/ 

Ps/faiM 

6/ycer& 

tfertr//? 

r/M//v 

Sto/v6 

&t?/ati/? 

+  39 

-.67 

-74 

-38 

'.93 

-79 

-.98 

/70rt//fy 

+.99 

-.66 

-.69 

-.99 

-/.00 

-.S3 

-/.00 

fn/o/ 

-67 

-.66 

+  .63 

-f.SO 

/.7/ 

+.33 

A7/ 

Pv/ate/ 

-.74 

-.69 

+  63 

+.66 

/.£? 

+.6Z 

+.73 

&/ycer0/ 

-.90 

-99 

+  .80 

+£6 

+.97 

+.90 

+.99 

0*itr/<0 

-.93 

-/.00 

-+.7/ 

+.65 

+.97 

+/.V0 

+/.00 

Inv//r? 

-.79 

-.63 

+.33 

+.02 

+.90 

+/.00 

+.96 

$rarcb 

-.9B 

-/.W 

+.7/ 

+.73 

+.99 

+-/.M 

+.06 

cent)  formed  indol  from  Witte's  peptone,  and  gas  was  formed  as 
follows:  sucrose  148  (98.2  per  cent) ;  raffinose  145  (96.2  per  cent) ; 
dulcitol  45  (29.8  per  cent) ;  glycerol  69  (45.6  per  cent) ;  salicin 
149  (98.8  per  cent');  dextrin  90  (59.5  per  cent);  inulin  22  (14.6 
per  cent)  and  starch  65  (43  per  cent) .  It  is  evident  from  table  1 
that  sucrose,  raffinose  and  salicin,  because  of  their  extreme 
availability,  cannot  be  employed  for  differentiation  within  the 
group.  The  coefficients  of  correlation  for  each  pair  of  remaining 
characters  are  given  in  table  IA. 

Mere  inspection  of  table  IA  shows  that  gelatin  liquefaction  is 
almost  perfectly  correlated  with  motility  and  fermentation  of 
glycerol,  dextrin,  and  starch;  the  association  being  positive  with 
motility  and  negative  with  the  others.  Similarly  motility  is 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP  261 

correlated  with  glycerol,  dextrin,  starch  and  gelatin.  Each  of 
these  characters  is  correlated  with  each  other.  Under  these  cir- 
cumstances any  of  these  reactions  may  be  selected  for  subdivision; 
the  choice  depending  upon  which  were  employed  in  an  investiga- 
tion and  to  some  extent  on  the  personal  preference  of  the  investi- 
gator. The  characterization  of  B.  aerogenes  by  Durham  as  a 
starch  fermenter;  the  differentiation  of  B.  aerogenes  from  B. 
cloacae  by  MacConkey  on  gelatin  liquefaction  and  motility, 
and  by  Kligler  on  glycerol  fermentation  are  all  correct;  the 
apparent  confusion  being  the  inevitable  result  of  separation  upon 
single  characters. 

Two  species  are  evidently  present,  the  B.  aerogenes  which 
rarely,  if  ever,  liquefies  gelatin;  is  non-motile;  and  forms  gas 
from  glycerol  and  starch;  and  the  B.  cloacae  which  liquefies 
gelatin  (often  very  slowly) ;  is  motile;  and  does  not  form  gas  from 
glycerol  nor  starch.  As  gelatin  liquefaction  is  an  inconvenient 
character  the  organisms  are  subdivided  for  further  study  upon 
motility  into  the  non-motile  B.  aerogenes  and  the  motile  B. 
cloacae.  Glycerol  or  starch  would  do  just  as  well.  Whichever 
character  is  selected,  a  few  strains  are  present  in  each  of  the  result- 
ing groups  which  possess  some  of  the  salient  characteristics  of  the 
other.  Thus  of  89  motile  strains  8  did  not  liquefy  gelatin,  8 
formed  gas  from  glycerol  and  4  from  starch,  while  of  62  non- 
motile  strains,  2  liquefied  gelatin,  and  glycerol  and  starch  were 
attacked  by  one. 

The  presence  of  a  few  supposedly  non-liquefiers  among  the 
motile  strains  may  as  probably — and  even  more  probably — be 
an  indication  of  the  inaccuracy  and  unreliability  of  the  gelatin 
liquefaction  test  than  of  the  presence  of  true  intermediate  organ- 
isms, for  the  number  of  gelatin  liquefiers  recognized  increases 
with  the  period  of  incubation.  Again  is  it  not  reasonable  to 
explain  the  presence  of  several  glycerol  and  starch  fermenters 
among  the  motile  strains  as  due  to  mixed  cultures?  Picking  off  a 
colony  from  a  plate,  even  after  several  replatings,  is  no  absolute 
criterion  that  a  pure  culture  was  obtained.  Some  species  stick 
tenaciously  together. 

One  of  the  motile  starch  fermenting  strains  referred  to  above 


262 


MAX   LEVINE 


was  plated  out  on  brilliant  green  agar.  Ten  colonies  were  fished 
into  motility  agar  and  starch;  three  were  non-motile  starch  fer- 
menters,  three  were  motile  and  did  not  attack  starch,  while  four 
were  both  motile  and  starch  fermenters  thus  indicating  that,  in 
this  instance  at  least,  the  presumably  overlapping  or  intermediate 

TABLE  2 
Showing  correlation  of  indol,  dulcitol,  and  inulin  for  62  strains  of  B.  aerogenes 


r/x/o/ 

M/fo/ 

Inutin  * 

y- 

-f- 

-f- 

1 

* 

3/ 

.20 

// 

7 

18 

- 

3/ 

/4 

/7 

u 

f 

1 

* 

20 

/4 

34 

8 

w 

- 

// 

17 

ZB 

/O 

/«^ 

1 

* 

7 

// 

d 

/O 

/8 

- 

/* 

/8 

20 

/6 

36 

Eight  cultures  not  tested  in  inulin. 

TABLE  2A 

Coefficient  of  correlation  for  each  pair  of  characters  in  table  2 


Indot 

Qu/cifo/ 

Inu/fn 

Indol 

+  .39 

-22 

Ov/cito/ 

+.39 

-£2 

Inif/in 

-.22 

-&e 

strains  are  in  all  probability  merely  mixed  cultures.  It  is  not 
contended  that  intermediate  strains  do  not  occur;  they  undoubt- 
edly do;  but  it  is  desired  to  point  out  that  these  have  been  over 
emphasized  in  the  past  and  that  the  plating  method  cannot  al- 
ways be  relied  upon  to  yield  pure  cultures. 

B.  cloacae  may  be  defined  as  a  gram  negative  short  rod  which 
ferments  lactose  weakly;  forms  acetylmethylcarbinol  from  glu- 
cose; is  alkaline  to  methyl-red;  motile;  rarely  forms  indol;  prac- 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP  263 

tically  always  forms  gas  from  sucrose,  raffinose,  mannitol  and 
salicin;  and  occasionally  from  dextrin;  gelatin  is  typically  liquefied; 
and  glycerol,  inulin  and  starch  are  not  fermented.  As  noted 
above,  the  few  glycerol,  inulin  and  starch  fermenters  are  probably 
due  to  mixed  cultures  and  may  be  dismissed. 

The  three  sucrose  negative  cultures  (also  raffinose  negative) 
may  be  regarded  as  a  variety  corresponding  to  the  B.  levans 
which  MacConkey  records  as  very  rare. 

The  dextrin  fermenters  probably  also  constitute  a  variety  of 
B.  cloacae  but  as  the  composition  of  dextrin  is  so  variable  we 
hesitate  to  employ  it  for  differential  purposes  for  the  present. 

B.  aerogenes  resembles  B.  cloacae  in  several  respects.  It  forms 
acetylmethylcarbinol  from  glucose;  is  alkaline  to  methyl-red; 
and  ferments  sucrose,  raffinose,  mannitol,  and  salicin  with  gas  for- 
mation. On  the  other  hand  lactose  is  more  vigorously  attacked; 
gelatin  is  typically  not  liquefied;  the  organisms  are  non-motile; 
while  glycerol  and  starch  are  fermented  with  gas  formation. 

Indol  was  formed  by  31  (50  per  cent);  gas  from  dulcitol  was 
formed  by  31  (50  per  cent) ;  and  from  inulin  by  18  (33  J  per  cent) 
of  the  B.  aerogenes  strains  (8  cultures  were  not  tested  with  inulin) . 
From  tables  2  and  2A  which  show  the  distribution  with  respect  to 
indol,  inulin  and  dulcitol,  and  the  correlation  coefficients  for  these 
reactions,  it  is  evident  that  the  characters  are  not  associated. 
They  may  be  of  significance  for  separation  of  varieties.  The 
utter  lack  of  correlation  necessitates  the  employment  of  all  of 
these  characters,  which  would  lead  to  the  formation  of  eight 
varieties.  It  is  deemed  unwise  to  establish  such  varieties  until 
more  extensive  collections  are  studied. 

THE   COLI   GROUP 

In  the  B.  coli  group  are  included  182  strains  quite  evenly  dis- 
tributed between  the  different  animal  sources,  sewage  and  soil. 
The  group  differs  sharply  from  the  B.  aerogenes-B.  cloacae  series 
in  that  the  Voges-Proskauer  reaction  is  negative  and  the  methyl- 
red  reaction  positive.  Starch  is  not  attacked  by  any  of  the 
strains.  It  has  been  shown  by  the  author  that  the  Voges-Pros- 


264 


MAX    LEVINE 


kauer  negative  strains  attack  the  monosaccharids  more  vigor- 
ously, but  the  disaccharids,  trisaccharid,  and  glucoside  no  less 
vigorously  than  the  Voges-Proskauer  positive  strains. 

In  table  3  is  shown  the  correlation  between  the  various  reac- 
tions.    As  all  strains  attacked  galactose,  lactose,  maltose  and 

TABLE  3 
Showing  correlation  of  characters  among  182  strains  of  the  coli  group 


MotililY 

Inc/ol 

Sucrose 

ffiff/wx 

Mcitol 

6/ycero/ 

Salicin 

+ 

— 

y-    — 

/- 

— 

-/-  \  — 

-/- 

— 

Y- 

— 

£ 

^ 

/ 

/SO 

//4\/6 

77 

S3 

77\53 

80 

50 

92 

37 

89 

41 

- 

\52 

49 

3 

/6 

36 

19  33 

17 

35 

33 

19 

25 

27 

|S 

/• 

//4\49 

163 

84 

79 

86 

77 

87 

76 

110 

52 

109 

54 

- 

/6   3 

\9 

9 

/O 

/O 

9 

/O 

9 

15 

4 

5 

/4 

1 

y- 

77\/6 

84  9 

95 

89 

4 

64 

29 

56 

36 

63 

30 

53 

36 

79/0 

39 

7 

82 

33 

56 

69. 

20 

51 

& 

1 

y- 

77/9 

86\/0 

89 

~7 

96 

65 

31 

60 

35 

66 

30 

- 

53  3d 

77\J_ 

4 

82 

86 

32 

54 

65 

21 

48 

38 

^ 

y- 

80\/7 

87 

10 

64 

33 

65 

52 

97 

63 

34 

75 

22 

50 

35 

76 

9 

29 

56 

3/ 

54 

85 

62 

22 

39 

46 

1 

/ 

92 

33 

I/O 

/5 

56 

69 

60 

65 

63 

62 

/25 

89 

36 

37 

19 

52 

4 

36 

20 

35 

2/ 

34 

22 

56 

25 

32 

I 

y- 

89 

25 

/09 

5 

63 

X 

66 

48 

75 

39 

89 

25 

//4 

- 

4/ 

?7 

54 

/4 

30 

38 

30 

38 

ZZ 

46 

36 

32 

68 

mannitol  with  gas  formation  and  failed  to  attack  the  polysac- 
•charids,  dextrin,  inulin  and  stafch,  or  to  liquefy  gelatin,  these  sub- 
stances are  not  included  in  the  table. 

The  proportion  of  positive  reactions  for  all  strains  of  the  B.  coli 
group  is  as  follows:  motility  130  (71.5  per  cent);  indol  163  (89.6 
per  cent) ;  sucrose  93  (51.1  per  cent) ;  raffinose  96  (52.7  per  cent) ; 
dulcitol  97  (53.3  per  cent);  glycerol  125  (68.8  per  cent)  and 
salicin  114  (62.7  per  cent). 

The  correlation  coefficients  for  each  pair  of  characters  is  given 
in  table  3A. 

The  highest  coefficient  obtained  for  motility  is  0.53  with  both 


CLASSIFICATION    OF    THE    COLON-CLOACAE    GROUP 


265 


sucrose  and  dulcitol.  Its  correlation  with  other  characters  is 
therefore  not  very  marked. 

Indol  seems  to  be  correlated  with  salicin,  but  the  small  propor- 
tion of  indol  negative  strains  (10.4  per  cent)  makes  the  associa- 
tion of  questionable  value,  and  as  the  coefficients  with  other 
substances  are  extremely  low,  indol  may  be  eliminated. 

Glycerol  correlates  somewhat  with  salicin  (coefficient  0.52)  but 

TABLE  SA 
Coefficients  of  correlation  for  each  pair  of  characters  in  table  3 


Wofi/ity 

f/xfo/ 

5ucrvse 

Fafffoax 

Dufcitvl 

Glycerol 

dal'icfn 

tfofility 

-39 

+.53 

+.43 

+.53 

+J8 

+.40 

Indot 

-.59 

+.08 

+.00 

+.02 

-.28 

+.76 

5(/crvx 

+.53 

+.08 

+.99 

+-.53 

-.38 

+.20 

Paffinax 

+.43 

+.OO 

+.99 

+.58 

-£9 

+.27 

Du/cito/ 

+.53 

+-.02 

+.56 

+.58 

-.21 

+-.60 

Glycero/ 

+.18 

-.28 

-.38 

-.29 

-.21 

+.52 

Salicio 

+.40 

+.76 

+.20 

+.27 

+.6O 

+.52 

with  no  other  character  and  is  therefore  not  considered  desirable 
for  subdivision  at  this  point. 

The  choice  of  a  differential  character  is  thus  narrowed  down 
to  sucrose,  raffinose,  dulcitol  and  salicin.  Sucrose  and  raffinose 
are  almost  perfectly  associated  (coefficient  of  correlation  0.99). 
Consideration  of  either  therefore  suffices  for  both  and  as  the 
former  is  slightly  better  correlated  with  other  characters,  sucrose 
is  selected  for  further  discussion. 

A  comparison  of  salicin  with  dulcitol  indicates  that  the  alcohol 
is  to  be  preferred.  Salicin  correlates  better  with  glycerol  and 
indol  (the  latter  relation  of  questionable  value),  while  dulcitol 
has  higher  coefficients  with  motility,  sucrose  and  raffinose.  A 
similar  consideration  leads  to  the  choice  of  sucrose  over  salicin. 


266  MAX    LEVINE 

Sucrose  and  dulcitol  therefore  remain.  These  are  the  two  char- 
acters in  regard  to  which  there  is  considerable  difference  of 
opinion  among  students  of  the  B.  coli  group.  MacConkey  gives 
preference  to  sucrose,  and  in  this  selection  is  supported  by  many 
investigators  (Howe  1912,  Kligler  1915,  Rogers  1915,  etc.); 
while  Jackson  (1911)  and  more  recently  Giltner  (1916)  subdivide 
first  on  dulcitol.  It  is  quite  interesting,  therefore,  that  on  the 
basis  of  the  correlation  coefficients  there  is  really  little  choice 
between  the  two.  Both  are  equally  well  correlated  with  motility 
(coefficient  0.53) ;  partially  with  each  other  (coefficient  0.58)  and 
not  associated  with  indol.  Dulcitol  correlates  partially  with 
salicin  (coefficient  0.60),  while  sucrose  does  not  (coefficient  0.20). 
On  the  other  hand,  sucrose  is  almost  perfectly  correlated  with 
raffinose  (coefficient  0.99),  whereas  salicin  is  only  partially  (co- 
efficient 0.58).  Although  neither  can  be  regarded  as  associated 
with  glycerol,  the  coefficient  with  sucrose  ( —0.38)  is  greater  than 
with  dulcitol  (-0.21). 

If  our  selection  is  to  be  guided  entirely  by  correlation,  the 
choice  between  dulcitol  and  sucrose  is  a  toss  up.  Sucrose  was 
finally  selected  for  the  primary  division  because  it  is  more 
widely  distributed  in  nature,  more  available  to  students  for 
investigational  purposes,  more  widely  accepted  by  bacteriologists, 
and  its  fermentation  better  correlated  with  the  source  than  is 
dulcitol  decomposition.  Differentiation  on  sucrose  yields  a 
sucrose  positive  group  of  93  strains,  and  a  sucrose  negative  group 
of  89  strains. 

THE  SUCROSE  NEGATIVE  STRAINS  OF  THE  COLI  GROUP 

Of  the  89  strains  which  did  not  form  gas  from  sucrose,  33 
(37.1  per  cent)  were  positive  in  dulcitol;  69  (77.6  per  cent)  posi- 
tive in  glycerol;  and  51  (57.3  per  cent)  gave  gas  in  salicin;  53 
(59.6  per  cent)  were  motile;  only  10  (11.3  per  cent)  failed  to  form 
indol. 

The  distribution  of  the  organisms,  with  regard  to  motility, 
dulcitol,  glycerol,  salicin  and  indol  is  given  in  table  4,  and  the 
coefficient  of  correlation  for  each  pair  of  reactions  is  given  in  table 
4A.  For  these  strains  motility  is  not  distinctly  correlated  with 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP 


267 


any  other  character.  Dulcitol  and  glycerol  are  not  correlated 
with  each  other,  nor  with  indol  and  motility,  but  each  has  a  high 
coefficient  of  association  with  salicin.  The  coefficient  for  dulcitol 

TABLE  4 
Showing  correlation  of  characters  among  89  sucrose  negative  strains  of  the  coli  group 


/ioff/ify 

Indol 

Ou/cHol 

6/ycerol 

5a//do 

•/- 

— 

•/- 

— 

•f- 

-t- 

+- 

1 

+ 

53 

45 

8 

22 

3/ 

43 

/O 

33 

20 

- 

56 

34 

2 

// 

25 

26 

/O 

/8 

18 

1 

/ 

45 

34 

79 

29 

50 

6i 

/8 

$/ 

28 

8 

2 

10 

4 

6 

8 

2 

10 

1 

j 

22 

// 

29 

4 

35 

27 

6 

28 

5 

- 

3/ 

25 

50 

6 

56 

42 

/4 

23 

33 

I 

+• 

43 

26 

£/ 

8 

<?7 

42 

69 

48 

T/ 

/O 

/O 

!6 

2 

6 

/* 

20 

3 

/7 

! 

/ 

33 

/6 

J/ 

28 

23 

48 

3 

5/ 

- 

20 

18 

28 

/O 

5 

33 

£V 

/7 

38 

TABLE  4 A 
Coefficients  of  correlation  for  each  pair  of  characters  in  table  4 


rtotffffy 

Zfrto/ 

0i/fata/ 

G/ycerv/ 

3a//a'/y 

/1oft//ty 

-.50 

+.22 

+  .25 

+  .25 

fndol 

-.50 

-.07 

-.08 

+/.00 

0v/afol 

+.22 

+.07 

+.SO 

+.78 

G/ycen/ 

+.£5 

-.08 

+.20 

+.86 

3  a/id  o 

t.25 

+/.00 

+.78 

+.86 

with  salicin  is  0.78  and  for  glycerol  with  salicin  is  0.86.  Indol  is 
also  correlated  with  salicin;  all  of  the  10  indol  negative  strains  are 
also  salicin  negative.  Differentiation  is  therefore  made  upon 
salicin  which  gives  a  sucrose  negative,  salicin  positive  subgroup 


268 


MAX    LEVINE 


of  51  strains,  and  a  sucrose  negative,  salicin  negative  subgroup  of 
38  organisms. 

The  sucrose-negative,  salicin-positive  subgroup  (B.  coli).  The 
distribution  of  the  51  sucrose  negative  salicin  positive  strains  on 
motility,  dulcitol,  and  glycerol  is  indicated  in  table  5.  33 
(64.9  per  cent)  are  motile;  28  (54.9  per  cent)  form  gas  from  dulci- 
tol, and  48  (94.3  per  cent)  from  glycerol.  The  extremely  small 
proportion  of  glycerol  negative  strains  (5.7  per  cent)  eliminates 


TABLE  5 


Showing  correlation  of  characters  among  51  sucrose  negative — salicin  positive  strains 
of  the  coli  group.     (B.  coli) 


/8 


15 


JO 


18 


to 


8 


/8 


18 


10 


Z8 


/5 


6 


Glycerol 


50 


/8 


£5 


Z3 


48 


*  Coefficient* of  correlation  for  motility  and  dulcitol  =  0.02. 

this  alcohol  from  further  statistical  consideration.  From  table 
5  it  is  seen  that  motility  and  dulcitol  are  not  correlated.  Further 
subdivision  on  correlated  characters  is  not  feasible.  This  entire 
group  then  is  regarded  as  the  species  B.  coli  and  two  varieties 
may  be  formed  on  motility — the  motile  B.  coli-communis  and  the 
non-motile  B.  coli-immobilis. 

The  sucrose  negative,  salicin  negative  subgroup  (B.  acidi-lactici) . 
Of  the  38  organisms  which  were  negative  for  both  sucrose  and 
salicin,  20  (52.7  per  cent)  were  motile,  28  (73.7  per  cent)  formed 
indol,  21  (55.3  per  cent)  were  positive  with  glycerol,  while  only 
5  (13.2  per  cent)  formed  gas  from  dulcitol  as  shown  in  table  6. 
From  table  6A  it  appears  that  motility  is  correlated  with  dulcitol 
fermentation  and  indol,  and  has  a  slightly  higher  coefficient  with 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP 


269 


glycerol  than  has  dulcitol.  Indol  has  a  slightly  higher  coefficient 
with  dulcitol  than  motility,  but  the  number  of  dulcitol  positive 
strains  is  so  small,  that  the  coefficients  observed  cannot  be  relied 
upon.  Indol  and  motility  seem  to  be  correlated  (coefficient 


TABLE  6 


Showing  correlation  of  characters  among  88  sucrose  negative,  salicin  negative  strains 
of  the  coli  group.     (B.  acidi-lactici} 


/odol 

flotilily 

Du/dtol 

G/ycero/ 

y- 

•/- 

-t- 

— 

-t- 

1 

/ 

<?£ 

tz 

/6 

2 

?6 

t4 

/4 

- 

to 

8 

2 

3 

7 

7 

3 

s 

/ 

A? 

8 

?0 

4 

/6 

/3 

7 

/6 

2 

/8 

/ 

/7 

& 

/O 

^ 

1 

/ 

2 

3 

4 

/ 

5 

2 

3 

- 

26 

7 

/6 

17 

35 

/9 

/4 

1 

1 

+ 

14 

7 

/3 

8 

2 

/? 

21 

- 

/4 

3 

7 

to 

3 

/4 

/7 

TABLE  6A 
Coefficients  of  correlation  for  each  pair  of  characters  in  table  6 


Indol 

Moti//ty 

Pu/ctfo/ 

G/ycero/ 

Indo/ 

+.68 

+.69 

+  .40 

/70////// 

+.68 

+.62 

+  .40 

0t//citol 

f.69 

+  .& 

+.34 

G/ycero/ 

+.40 

+.40 

+.34 

0.68),  and  their  coefficients  with  glycerol  are  identical  (0.40). 
In  a  preliminary  report  subdivision  was  made  upon  motility  into 
the  motile  species  B.  Grwnthal,  and  non-motile  B.  acidi-lactici. 
It  seems  best,  until  more  extensive  collections  are  studied,  that 


270 


MAX    LEVINE 


all  of  the  sucrose-negative,  salicin-negative  strains  be  included 
in  the  species  B.  acidi-lactici  in  which  may  be  recognized  two 
varieties,  the  motile  B.  acidi-lactici  var.  Gruenthali  and  the  non- 
motile  B.  acidi-lactici  var.  immobili. 

TABLE  7 
Showing  correlation  of  characters  among  93  sucrose  positive  strains  in  the  coli  group 


Woi 

Wfy 

I/3C/0/ 

G/yt 

#rd 

50//t 

~isy 

j£ 

77 

69 

8 

58 

/9 

49 

27 

56 

2/ 

^~ 

/6 

/5 

/ 

6 

/O 

7 

9 

7 

9 

V 

69 

/5 

84 

58 

26 

49 

34 

58 

£6 

1: 

8 

/ 

9 

6 

3 

7 

2 

5 

4 

I71 

58 

6 

58 

6 

64 

36 

Z8 

47 

/7 

1- 

/9 

/O 

26 

3 

29 

20 

8 

/6 

0 

\l 

49 

7 

49 

7 

36 

20 

56 

41 

/5 

^>- 

27 

9 

34 

2 

26 

8 

36 

2/ 

/5 

i' 

56 

7 

58 

5 

47 

/6 

4/ 

2/ 

63 

s- 

2/ 

9 

26 

4 

/7 

/J 

(5 

/5 

30 

TABLE  7x 
Coefficients  of  correlation  for  each  pair  of  characters  in  table  7 


tfon/ity 

Itttol 

Ov/citol 

G/yctrd 

5a/fcio 

r?otility 

-.27 

+.67 

+.40 

+.54 

Indol 

-.?7 

+.05 

-.42 

+.Z8 

M/fo/ 

+.67 

+.05 

-.32 

+.39 

6/ycero/' 

+.40 

-.42 

-.32 

+.32 

Safari 

+.54 

+.Z8 

+.39 

+.32 

THE   SUCROSE    POSITIVE   STRAINS   OF   THE    COLI    GROUP 

Of  the  93  organisms  which  fermented  sucrose  with  gas  forma- 
tion, 77  (82.8  per  cent)  were  motile;  84  (90.4  per  cent)  formed 
indol;  and  positive  gas  reactions  were  obtained  as  follows: 
dulcitol  64  (72.1  per  cent);  glycerol  56  (60.2  per  cent)  and  salicin 


CLASSIFICATION   OF   THE    COLON-CLOACAE    GROUP 


271 


63  (67.8  per  cent).  The  distribution  with  respect  to  these  char- 
acters and  the  correlation  coefficients  are  shown  in  tables  7  and 
7A  respectively,  where  it  is  seen  that  motility  correlates  better 
with  dulcitol  than  does  any  other  of  the  characters.  It  also 
correlates  best  with  salicin.  Motility  is  the  best  correlated 

T-ABLE  8 

Showing  correlation  of  characters  among  77  sucrose  positive  motile  strains  of  the  coli 

group.     (B.  communior) 


Itx/ot 


69 


/6 


/8 


M/M 


53 


58 


26 


41 


/7 


/6 


19 


32  f  7 


G/ycero/  Salicin 


32 


17 


49 


J7 


25 


26 


27 


/9 


37 


17 


8 


2/ 


TABLE  SA 
Coefficients  of  correlation  for  each  pair  of  characters  in  table 


Zrxfo/ 

0otito/ 

G/ycero/ 

5a/ic/a 

£rto/ 

+.33 

-.?7 

+^6 

M/fo/ 

+.33 

-.87 

-.sz 

tf/ycew/ 

-.e7 

-.67 

+.O9 

5a//c/n 

+.S6 

-£S 

+.09 

character.  There  are  thus  two  subgroups,  a  sucrose-positive, 
motile  subgroup  of  77  strains  and  a  sucrose  positive  non-motile 
subgroup  comprising  16  strains. 

The  sucrose  positive  motile  subgroup  (B.  communior).  Inspec- 
tion of  tables  8  and  SA  shows  that  among  the  sucrose  positive 
motile  strains,  neither  indol  production  nor  salicin  fermentation 
is  correlated  with  other  characters.  Gas  formation  from  dulcitol 


272 


MAX    LEVINE 


and  glycerol  shows  a  strong  negative  association.  Those  strains 
which  failed  to  attack  glycerol  practically  always  fermented 
dulcitol.  Thus  26  of  27  glycerol  negative  are  dulcitol  positive 
while  17  of  18  dulcitol  non-fermenters,  tested,  formed  gas  from 
glycerol.  To  put  it  another  way,  inability  to  attack  either  of 
the  alcohols  is  accompanied  by  fermentation  of  the  other.  Fer- 
mentation of  either,  however,  yields  but  little  information  as  to 

TABLE  9 

Showing  correlation  of  characters  among  16  sucrose  positive,  non-motile  strains,  of 

the  coli  group 


0V/C 

to/ 

6/y 

•^era/ 

5<lf< 

tin 

J- 

6 

4 

£> 

6 

•t 
2; 

/O 

3 

7 

/ 

9 

V 

4 

3 

7 

5 

2 

1- 

2 

7 

9. 

Z 

7 

*- 

6 

/ 

5 

2 

7 

1- 

9 

^ 

-7 

9. 

TABLE  9A 
Coefficients  of  correlation  for  each  pair  of  characters  in  table  9 


0uM 

G/ycerv/ 

5a//&n 

DV/C//O/ 

+.65 

+  /.00 

G/yceroJ 

+.65 

+  .80 

5a//cfo 

+AOO 

+.80 

how  the  other  will  react.  The  desirability  of  subdividing  on 
either  glycerol  or  dulcitol  to  form  two  species  is  questioned.  For 
the  present  the  entire  group  of  sucrose  fermenting  motile  forms  is 
designated  as  B.  communior  and  two  varieties  may  be  formed  on 
glycerol  or  dulcitol. 

The  sucrose-positive  non-motile  subgroup.  Only  16  of  the 
sucrose  fermenters  were  non-motile,  and  only  one  of  these  failed 
to  produce  indol.  Although  the  number  of  organisms  is  small,  it 
is  quite  surprising  that  they  should  be  so  evenly  divided  with 
respect  to  gas  formation  from  the  test  substances.  Thus  6 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP 


273 


(37.5  per  cent)  are  positive  with  dulcitol,  7  (43.7  per  cent)  with 
glycerol,  and  7  (43.7  per  cent)  with  salicm.  From  tables  9  and 
QA  it  is  seen  that  salicin  is  the  best  correlated  character.  Of  the 
seven  salicin  positive  strains,  6  (85.8  per  cent)  attack  dulcitol  and 
5  (71.5  per  cent)  glycerol.  The  characteristics  of  this  group 

X 

TABLE  10 

Distribution  of  organisms  from  different  sources  among  the  various  species  and 

varieties 


3,  c/oa- 
C.oe 

8.  aero- 
genes 

B.com- 
munior 

B.netpol- 
ifanus 

B.COS- 

coroba 

B.ct 

//' 

B.ocidi-lactici 

Ton/ 

commas 

'mmeWis 

JFUGftlholi 

immobili 

So!/ 

ff° 

68 

54 

£6 

O 

0 

2 

0 

7 

0 

177 

% 

43.7 

30.5 

14.7 

U 

4.0 

Horse 

Uo 

O 

0 

15 

0 

O 

4- 

O 

0 

O 

P 

% 

79.0 

2/.0 

Sheep 

No 

0 

0 

16 

0 

5 

1 

0 

0 

0 

ZZ 

% 

72.8 

2Z.7 

4.5 

Cow 

No 

O 

0 

6 

4 

0 

9 

O 

/ 

0 

ZQ 

% 

30.0 

20.0 

45.0 

5.0 

p,9 

Wo 

O 

O 

9 

0 

/ 

// 

/ 

9 

O 

31 

% 

29.0 

3£ 

55.6 

3£ 

29.0 

5ewaqe 

Mo 

1 

8 

3 

3 

z 

/ 

IE 

2 

7 

39 

% 

2.6 

Z0.5 

7.7 

7.7 

5J 

2.6 

30.8 

5.1 

17.9 

Kan 

fYo 

0 

O 

2 

0 

/ 

5 

5 

/ 

II 

25 

% 

8.0 

4.0 

20.0 

£0.0 

4.0 

44.0 

Total 

d? 

62 

77 

7 

9 

33 

16 

20 

18- 

333 

therefore  resemble  the  E.  neapolitanus  of  MacConkey's  varieties. 
On  the  other  hand,  7  (77.8  per  cent)  of  the  9  salicin  negative 
strains  are  negative  for  glycerol  while  all  failed  to  ferment  dulcitol. 
These  are  therefore  the  B.  coscoroba  of  MacConkey's  classification. 

RELATION   OF   SPECIES   TO    SOURCE 

Table  10  shows  the  distribution  of  the  organisms  from  different 
sources  among  the  various  species  and  varieties.  Species  or 
varieties  and  habitat  seem  to  be  somewhat  related. 

B.  aerogenes  and  B.  cloacae  were  obtained  only  from  soil  and 
sewage  and  were  not  isolated  from  any  of  the  animals  tested. 
B.  cloacae  constituted  49.7  per  cent  of  the  soil  and  2.6  per  cent 


THE  JOURNAL  OF  BACTERIOLOGY,  VOL.  Ill,  NO.  3 


274  MAX    LEVINE 

of  the  sewage  strains,  while  30.5  per  cent  from  soil  and  20.5  per 
cent  from  sewage  were  B.  aerogenes. 

B.  communior  was  isolated  from  all  sources  as  follows:  soil 
14.7  per  cent;  horse  79  per  cent;  sheep  72.8  per  cent;  cow  30  per 
cent;  pig  29  per  cent;  sewage  7.7  per  cent  and  man  8  per  cent. 
The  relative  abundance  of  B.  communior  among  the  lower  animals 
and  scarcity  in  man  and  sewage,  may  well  be  investigated  further. 

B.  neapolitanus  was  present  only  in  bovine  feces  and  sewage, 
comprising  20  per  cent  of  the  bovine  and  7.7  per  cent  of  the 
sewage  strains. 

Of  the  9  B.  coscoroba,  5  were  from  sheep,  1  from  pig,  2  from 
sewage,  and  1  from  man.  22.7  per  cent  of  sheep,  3.2  per  cent 
of  pig,  5.1  per  cent  of  sewage  and  4  per  cent  of  human  strains  fall 
in  this  species. 

B.  coliy  like  B.  communior  was  isolated  from  all  of  the  sources 
tested,  but  a  rather  distinct  correlation  with  the  source  is  ob- 
served with  the  varieties  B.  coli-communis  and  B.  coli-immobilis. 
The  former  comprise  1.1  per  cent  of  soil;  21  per  cent  of  horse; 

4.5  per  cent  of  sheep;  45  per  cent  of  cow;  35.6  per  cent  of  pig; 

2.6  per  cent  of  sewage;  and  20  per  cent  of  human  strains.     B.  coli- 
immobilis  was  not  obtained  from  the  soil,  horse,  sheep  or  cow,  but 
it  made  up  3.2  per  cent  of  the  pig,  30.8  per  cent  of  the  sewage, 
and  20  per  cent  of  the  human  strains. 

B.  acidi-lactici  was  not  obtained  from  the  horse  nor  sheep, 
and  only  rarely  from  the  cow  (5.  per  cent)  or  soil  (4  per  cent). 
The  motile  variety  B.  acidi-lactici  var.  Gruenthali  was  particularly 
abundant  among  the  pig  cultures  (29  per  cent)  and  rare  in  sewage 
(5.1  per  cent)  and  man  (4  per  cent).  The  non-motile  B.  acidi- 
lactici  far.  immobili  Was  restricted  to  man  and  sewage  entirely, 
comprising  44  per  cent  of  the  human  and  17.9  per  cent  of  the 
sewage  strains. 

If  subsequent  and  more  extensive  studies  confirm  these  results 
the  determination  of  species  and  varieties  would  have  some  bear- 
ing on  the  interpretation  of  the  colon  test. 

The  author  takes  this  opportunity  to  express  his  gratitude  to 
Dr.  R.  E.  Buchanan  for  many  helpful  suggestions  and  encour- 
agement, and  to  Prof.  G.  W.  Snedecor  for  assistance  and  eluci- 
dation of  the  mathematical  principles  involved. 


CLASSIFICATION    OF   THE    COLON-CLOACAE    GROUP 


275 


SUMMARY 


From  a  statistical  study  of  333  coli-like  bacteria  isolated  from 
soil,  sewage,  and  the  feces  of  various  animals,  the  following 
classification  is  suggested: 


Lactose  y- 


>r~oper?e£ 


Co//' 


5f  arcfr 


S70ft//fy- 


Sacrose- 


&.  c/c&cae 


j3. 


0.  co/i 


3a/ic/'>?  -f-        Sa/ic/'a  - 


*  Designation  as  species  questionable.     Probably  preferable  to  regard  it  as  a 
variety  of  B.  communior. 

TABLE  11 
Per  cent  of  positive  reactions 


V.P. 

fab/ 

Ge/a/?0 

ffofi/fy 

5fort£ 

Jar/fa 

PexfriD 

fr/Mb 

/&ffiha>i 

Jvcrax 

fl//afo/ 

G/yce/d 

Watf 
3mm& 

B.c/oacae 

/O  0.0 

/6.8 

9/.0 

/OO.O 

4.5 

4.5 

30.4 

98.0 

?3.3 

%.7 

&7 

9.0 

89 

6.a&v^eoK 

/00*0 

50.0 

3.2 

0.0 

98.5 

Z9J 

/OO.O 

/OO.O 

/00.0 

/00.0 

50.0 

98.5 

62 

B.ccmrwriw 

0.0 

89.6 

0.0 

/OO.O 

o.o 

0.0 

0.0 

7Z8 

94.8 

/OO.O 

75.4 

63.7 

77 

B.f%0f>o//toxo 

0.0 

/00.0 

0.0 

0.0 

0.0 

0.0 

0.0 

/00.0 

/OO.O 

fOO.O 

35.8 

7/.5 

7 

fi.CQ5carnfa 

0.0 

89.0 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

/00.0 

/OO.O 

0.0 

222 

9 

B.cot; 

p—  ^—  —  -^«—  ^ 

8iaciiffto& 

0.0 
0.0 

/OO.O 
640 

0.0 
0.0 

64.7 
5?7 

0.0 
0.0 

0.0 
0.0 

0.0 
0.0 

/W.O 
0.0 

//.& 
2.6 

0.0 
0.0 

55£ 

ft? 

94.3 
55.Z 

J/ 

38 

*  Ten  questionable  reactions  included. 

The  per  cent  of  positive  reactions  of  the  different  species  is 
indicated  in  table  11.     B.  neapolitanus  differs  from  B.  communior 


276  MAX   LEVINE 

only  with  respect  to  motility,  and  it  may  therefore  be  well  to 
regard  it  as  a  variety  of  B.  communior.  However  B.  coscoroba 
is  so  distinctly  different  from  the  other  sucrose  fermenters  that  its 
designation  as  a  species  seems  justified.  It  should  be  borne  in 
mind  however,  that  the  differentiation  in  this  instance  is  based 
on  only  9  individual  cultures  so  that  the  correlations  observed 
must  not  be  over  emphasized. 

CONCLUSIONS 

To  treat  all  characters  as  of  equal  taxonomic  significance  leads 
to  an  infinite  number  of  unstable  varieties;  a  condition  to  be 
avoided. 

Subdivision  on  correlated  characters  results  in  a  small  number 
of  groups  which  possess  considerable  stability. 

The  species  described  are  quite  strikingly  correlated  with  the 
source,  and,  if  more  extensive  investigations  confirm  these  ob- 
servations, recognition  of  the  various  species  may  be  of  sanitary 
significance. 

It  is  not  supposed  that  the  classification  presented  is  the  last 
word  in  the  differentiation  of  coli-like  bacteria,  but  it  is  hoped 
that  if  subdivision  is  to  be  made  upon  correlated  characters — and 
there  is  much  to  commend  such  a  procedure — the  method  de- 
scribed in  this  paper  for  the  determination  of  the  best  correlated 
character,  by  a  study  of  the  coefficients  of  correlation,  will  be  an 
aid  to  later  investigators. 

REFERENCES 

BERGEY  AND  DEEHAN,  S.  J.     1908    J.  Med.  Research,  19,  175. 

DURHAM,  H.  E.     1901    J.  E^er.,  5,  353. 

FORD,  W.  W.     1903    Studies  from  the  Rockefeller  Inst.  of  Med.  Res.  11. 

HOWE,  E.  C.     1912    Science,  N.  S.,  35,  225. 

JACKSON,  D.  D.     1911    Am.  J.  Pub.  Health,  1,  930. 

JOHNSON  AND  LEVINE    1917    J.  Bact. 

KLIGLER,  I.  J.,     1914    J.  Infect.  Dis.,  15,  135. 

LEVINE,  M.     1916    J.  Infect.  Dis.,  19,  773. 

MAcCoNKEY,  A.     1905    J.  Hyg.  5,  333;  1909    J.  Hyg.  9. 

ROGERS,  ET  AL.     J.  Infect.  Dis.,     1914,  14,  411;  1914,  15,  100;  1915,[l7,^137. 

SMITH,  TH.     1893    The  Wilder  Quarter  Century  Book.     187. 

YULE,  1916    An  introduction  to  the  theory  of  statistics. 


Differentiation  of  B.  Coli  and  B.  Aerogenes 

on  a  Simplified  Eosin-Methylene 

Blue  Agar 


MAX     LEVINE 


Reprinted  from 
THE  JOURNAL  OF  INFECTIOUS  DISEASES,  Vol.  23,  No.  1,  July,  1918,  pp.  43-47 


DIFFERENTIATION   OF  B.   COLI  AND  B.  AEROGENES  ON 
A  SIMPLIFIED  EOSIN-METHYLENE  BLUE  AGAR 

MAX    LEVINE 

From  the  Laboratory  of  the  Department  of  Pathology  and  Bacteriology,  State  University  of 

Iowa,  Iowa  City,  Iowa 

For  confirming  the  presumptive  test  for  B.  coli  the  mediums  most 
frequently  employed  are  litmus  lactose  agar  and  fuchsin  sulphite 
(Endo)  agar.  It  is  becoming  more  apparent  that  the  coli-like  forms 
may  be  divided  into  two  groups  which  are  closely  correlated  with  the 
source.  One  group  (B.  coli)  is  characteristic  of  fecal  origin;  the 
other  (B.  aerogenes  and  B.  cloacae)  is  rare  in  feces,  but  constitutes 
the  prevailing  coli-like  form  in  the  soil  and  on  grains.  The  standard 
litmus  lactose  and  Endo  agar  may  be  employed  to  a  slight  extent  for 
the  differentiation  of  B.  coli  and  B.  aerogenes,  but  the  differences 
between  these  types  on  these  mediums  (particularly  L.L.A.)  are  not 
very  clear-cut  nor  distinct.  Better  results  are  obtained  with  a  modified 
Endo  agar  described  elsewhere.  A  very  excellent  differentiation 
between  the  B.  coli  and  B.  aerogenes  types  has  been  obtained  on  a 
modification  of  eosin-methylene  blue  agar  first  described  by  Holt- 
Harris  and  Teague  for  the  isolation  of  the  typhoid  group  from  feces. 
The  medium  is  prepared  in  the  following  manner : 

Distilled  water  1000  cc 

Peptone  (Difco)  10  gm. 

Dipotassium  phosphate  2  gm. 

Agar  15  gm. 

Boil  ingredients  until  dissolved  and  make  up  any  loss  due  to  evaporation. 
Place  measured  quantities  in  flasks  and  sterilize  at  15  Ibs.  for  15  minutes. 
Just  prior  to  use  add  to  each  100  cc  of  the  melted  agar,  prepared  as  above, 
the  following  constituents: 

Sterile  (20%)  lactose  solution  1  gm.  or  5  cc 

Aqueous   (2%)  eosin   (yellowish)   solution        2  cc 
*_ Aqueous  rf%)   methylene  blue  solution  2  cc 


Pour  medium  into  petri  dishes,  allow  them  to  harden  in  incubator  and 
inoculate  in  the  ordinary  way.  Smearing  the  surface  with  a  glass  rod  seems 
preferable  to  the  streaking  method  sometimes  employed. 

There  is  no  adjustment  of  reaction  and  filtration  of  medium  is  not  necessary.. 

B.  typhosus  and  members  of  intermediate  group  also  grow  well  on 
this  medium  producing  transparent,  colorless,  or  slightly  amber  colonies 
that  are  about  one-half  the  size  of  B.  coli. 

Received  for  publication  Feb.  7,  1918. 


MAX  LEVINE 


DIFFERENTIATION   OF   B.    COLI   AND   B.   AEROGENES   ON   EOSIN-METHYLENE 

BLUE    AGAR 


Size: 


Confluence : 


Elevation : 


Appearance     by 
mitted  Light : 


Trans- 


Appearance  by  Reflected 
Light : 


B.  coli 

Well  isolated  colonies 
are  3-4  mm.  in  di- 
ameter. 

Neighboring  colonies 
show  little  tendency  to 
run  together. 

Colonies  slightly  raised; 
surface  flat  or  slightly 
concave,  rarely  convex. 


Dark  almost  black  cen- 
ters which  extend 
more  than  %  across 
the  diameter  of  col- 
ony ;  internal  struc- 
ture of  central  dark 
portion  difficult  to  dis- 
cern. 

Colonies  dark,  button- 
like,  often  concentric- 
ally ringed  with  a 
greenish  metallic  sheen. 


B.  aerogenes. 
Well     isolated     colonies 
are    larger    than    coli ; 
usually  4-6  mm.  in  di- 
ameter or  more. 

Neighboring  colonies  run 
together  quickly. 

Colonies  considerably 
raised  and  markedly 
convex ;  occasionally 
the  center  drops  pre- 
cipitately. 

Centers  deep  brown ;  not 
as  dark  as  B.  coli,  and 
smaller  in  proportion 
to  the  rest  of  the 
colony.  Striated  inter- 
nal structure  often  ob- 
served in  young  col- 
onies. 


Much  lighter  than  B. 
coli..  Metallic  sheen 
not  observed  except 
occasionally  i  n  de- 
pressed center  when 
such  is  present. 

RESULTS     WITH     PURE     CULTURES 

A  number  of  pure  cultures  were  employed  to  test  the  value  of  this 
medium  for  the  differentiation  of  B.  coli,  B.  aerogenes,  and  members 
of  the  typhoid  and  paratyphoid  groups. 

Of  22  cultures  of  B.  aerogenes  all  but  3  gave  the  characteristic 
reactions.  Of  these  3  cultures,  1  resembled  B.  coli  on  the  eosin- 
methylene  blue  agar,  another  failed  to  produce  a  black  center,  and  the 
3rd  showed  a  slight  metallic  lustre,  but  did  not  resemble  B.  coll  closely. 

Of  35  cultures  of  B.  coli  tested,  29  were  typical.  Six  did  not  show 
a  distinct  metallic  lustre,  but  were  typical  in  other  respects. 

There  were  23  strains  of  B.  cholerasuis,  B.  paratyphosus,  and 
B.  typhosus  tested.  One  strain  of  B.  paratyphosus  A  did  not  grow. 
All  other  strains  of  the  intermediate  group  developed  typical  trans- 
parent colonies. 

RESULTS    OBTAINED    WITH    WATER    SAMPLES 

The  differentiation  of  pure  strains  seemed  to  be  so  marked  and 
distinct  that  it  was  thought  the  medium  might  be  employed  for  con- 
firmation of  the  presumptive  test  for  B.  coli,  and  that  it  might  be  pos- 


DIFFERENTIATION  OF  B.  COLI  AND  B.  AEROGENES  5 

sible  to  differentiate  B.  coli  from  B.  aerogenes  simultaneously  with 
confirming  the  presumptive  test.  For  this  purpose  the  following 
experiment  was  carried  out. 

Seven  samples  of  water  from  different  parts  of  the  Iowa  river,  one  of 
sewage,  one  of  a  small  creek,  and  one  from  a  stagnant  body  of  water  were 
plated  out  directly  on  litmus  lactose  agar  and  inoculated  into  lactose  broth. 
After  24  hours'  incubation  10  acid  colonies  were  fished  from  the  litmus  lactose 
agar  plates  of  each  sample  for  further  study.  The  lactose  broth  tubes  were 
plated  out  after  48  hours'  incubation  onto  eosin-methylene  blue  agar  and  onto 
litmus  lactose  agar.  After  24  hours'  incubation  10  colonies  were  fished  from 
these  litmus  lactose  agar  plates  of  cash  sample  for  further  observation.  From 
the  eosin-methylene  blue  plates  made  from  the  preliminary  lactose  broth  tubes 
colonies  which  resembled  B.  coli  or  B.  aerogenes  were  fished  and  tentatively 
designated  as  such,  with  a  view  to  determining  the  accuracy  and  reliability  of 
the  plate  differentiation. 

All  colonies  fished  from  litmus  lactose  agar  were  reinoculated  into  lactose 
broth  and  after  24  hours'  incubation  were  plated  out  on  eosin-methylene  blue 
agar.  From  each  plate  was  picked  a  well  isolated  colony  which  was  designated 
as  B.  coli  or  B.  aerogenes.  These  designations  were  then  checked  by  growing 
the  organisms  in  Clark  and  Lubs  medium  and  testing  with  the  methyl-red  and 
Voges-Proskauer  reactions. 

CULTURES    OBTAINED    DIRECTLY    FROM    'LITMUS    LACTOSE    AGAR 

Of  the  10  water  samples  examined  1  did  not  show  acid  colonies  by 
direct  plating  on  litmus  lactose  agar.  Of  the  90  acid  colonies  fished, 
3  were  found  to  be  lactose  nonfermenters.  Thirty-three  cultures  were 
regarded,  from  their  appearance  on  eosin-methylene  blue  agar,  as  B. 
aerogenes.  Of  these,  24  (72%)  gave  the  Voges-Proskauer  reaction. 
Seven  cultures  were  diagnosed  tentatively  as  questionable  but  probably 
B.  aerogenes  but  none  of  these  gave  a  Voges-Proskauer  reaction. 

Eight  cultures  were  regarded  as  questionable,  but  probably  B.  coli, 
of  which  6  (75%)  did  not  give  the  Voges-Proskauer  reaction.  Thirty- 
nine  cultures  were  designated  from  their  appearance  on  eosin-methy- 
lene blue  agar,  as  B.  coli,  and  all  were  confirmed,  as  none  gave  the 
Voges-Proskauer  reaction. 

Of  40  cultures  which  were  regarded  tentatively  as  B.  aerogenes  or 
probably  B.  aerogenes  24  (60%)  were  correct.  Of  47  cultures  regarded 
as  B.  coli  or  probably  B.  coli,  45  (95.8%)  were  correct. 

CULTURES   OBTAINED   FROM    LITMUS   LACTOSE   AGAR   AFTER   PRELIMINARY 
ENRICHMENT    IN    LACTOSE    BROTH 

After  elimination  of  a  few  strains  which  proved  to  be  other  than 
coli  forms,  85  cultures  which  were  isolated  from  litmus  lactose  agar 
plates  made  from  the  lactose  broth  preliminary  enrichment  tubes  were 


6  MAX  LEVINE 

smeared  onto  eosin-methylene  blue  agar  for  differentiation.  One 
organism  was  regarded  as  probably  B.  aerogenes  and  on  confirmation 
proved  to  be  B.  coli.  Of  35  organisms  recorded  as  B.  aerogenes  34 
(97%)  gave  the  Voges-Proskauer  reaction.  Forty-nine  organisms 
were  tentatively  designated  as  B.  coli  or  probably  B.  coli  and  all 
proved  to  be  negative  for  the  Voges-Proskauer  reaction. 

With  organisms  isolated  from  this  group  then,  34  out  of  36 
cultures  regarded  as  B.  aerogenes  were  confirmed  as  such  while  every 
one  of  49  strains  regarded  as  B.  coli  was  correct. 

CULTURES      OBTAINED      FROM      EOSIN-METHYLENE      BLUE      AGAR      AFTER 
PRELIMINARY    ENRICHMENT    IN    LACTOSE    BROTH 

Fifty-two  cultures  were  fished,  26  of  which  were  regarded  as 
B.  aerogenes  and  the  remaining  as  B.  coli.  Of  the  26  supposedly 
B.  aerogenes  strains  all  gave  the  Voges-Proskauer  reaction.  Two  of 
the  strains  regarded  as  B.  coli  also  gave  the  Voges-Proskauer  reaction. 
Thus  100%  of  the  B.  aerogenes  strains  and  92.4%  of  the  B.  coli 
strains  were  correctly  differentiated  on  eosin-methylene  blue  agar. 

Results  on  all  cultures  isolated  may  be  summarized  as  follows : 

Tentatively  regarded  as  B.  coli  from  appearance  of  eosin-methy- 
lene blue  agar 122 

Correctly  designated  as  indicated  by  negative  Voges-Proskauer 
reaction 118 

Per  cent,  confirmed 96.9 

Tentatively  regarded  as  B.  aerogenes  from  appearance  on  eosin- 
methylene  blue  agar 102 

Correctly  designated  as  indicated  by  positive  Voges-Proskauer 
reaction 84 

Per   cent,    confirmed 82.4 

CORRELATION     OF     VOGES-PROSKAUER     AND     METHYL-RED     REACTION 

In  previous  work  a  marked  correlation  was  observed  between  the 
Voges-Proskauer  and  methyl-red  reactions.  Strains  of  coli-like  forms 
which  were  acid  to  methyl-red  characteristically  did  not  give  a  test 
for  acetyl-methyl-carbinal  (V-P  negative)  ;  while  those  reacting  alka- 
line to  methyl-red  gave  a  positive  Voges-Proskauer  test.  These 
observations  were  confirmed  by  Greenfield,1  Hunter,2  Clark,3  Hutton,4 
Rettger  and  Burton5  and  others. 


DIFFERENTIATION  OF  B.  COLI  AND  B.  AEROGENES  7 

In  this  group  of  strains  studied  a  similar  correlation  was  observed. 
The  relation  between  the  Voges-Proskauer  and  methyl-red  reactions  is 
indicated  in  the  following  table : 

Methyl-red 

+    '  N*  — 

°?+  2  2  84 

>  —  121  13  2 

*  In   previous   work   neutral   reactions   to   methyl-red    have    been    grouped    with    the    acid 
strains. 

It  is  seen  from  the  table  that  there  is  an  excellent  correlation 
between  the  two  reactions.  Placing  the  neutral  reacting  strains  with 
the  acid  group  as  was  done  in  previous  studies,  we  find  that  84 
(97.8%)  of  86  methyl-red  negative  strains  give  the  V.  P.  reaction; 
while  of  136  strains  not  giving  the  V.  P.  reaction  134  (98.7%)  were 
acid  or  neutral  to  methyl-red. 

In  this  series  of  cultures  the  V.  P.  reactions  were  more  clear-cut 
than  the  methyl-red  test  but  we  have  worked  with  collections  in  which 
the  reverse  was  true.  It  seems  best  to  employ  both  the  V.  P.  and 
methyl-red  tests  and  to  repeat  if  the  results  do  not  agree. 

It  is  interesting  to  note  that  of  87  cultures  isolated  from  litmus 
lactose  agar  plates  made  directly,  29.9%  proved  to  be  B.  aerogenes; 
whereas  of  85  cultures  isolated  from  litmus  lactose  agar  plates  made 
after  preliminary  enrichment  for  48  hours  in  lactose  broth,  40%  proved 
to  be  B.  aerogenes.  "This  indicates  the  correctness  of  the  conten- 
tion of  Race  and  others  that  preliminary  enrichment  tends  to  an  over- 
growth of  B.  aerogenes  types. 

Organisms  other  than  B.  coli  and  B.  aerogenes  grow  quite  well  on 
this  medium  and  several  have  been  observed  to  produce  small  blue 
centers ;  but  the  appearance  is  so  distinct  from  B.  coli  and  B.  aerogenes 
that  once  having  observed  the  true  types  there  should  be  no  mistake. 
Just  what  these  other  forms  are  has  not  been  determined  but  several 
have  been  isolated  and  will  be  reported  on  in  a  future  report.  They 
produce  very  small  colonies  with  pinpoint  light  blue  or  delf-blue  cen- 
ters. The  color  is  very  different  from  the  brownish  black  appearance 
of  the  B.  coli  and  B.  aerogenes  types.  Perhaps  the  introduction  of 
some  inhibitory  dye  into  the  medium  will  make  it  even  more  reliable 
.•for  the  isolation  and  differentiation  of  B.  coli  and  B.  aerogenes  and 
confirmation  of  the  presumptive  test. 

Jour.   Infect.  Dis.,   1916,   19,  p.   647. 
Jour.  Bacteriol.,  1917,  2,  p.   585. 
Jour.  Biol.   Chem.,   1917,   30,   p.   209. 
Jour.  Infect.  Dis.,  1916,   19,  p.  606. 
Ibid.,  21,  p.   162. 


Dysentery  and  Allied  Bacilli 


MAX     LEVINE 


Reprinted  from 
THE  JOURNAL  OF  INFECTIIOUS  DISEASES,  Vol.  27,  No.  1,  July,  1920,  pp.  31-39 


DYSENTERY    AND     ALLIED     BACILLI 

MAX    LEVI  N  E 

From  the  Laboratory  of  the  Central  Medical  Department,  A.  E.  F.,  Dijon,  France,  and  the 
Army  Medical  School,    Washington,  D.   C. 

In  France  we  not  infrequently  experienced  difficulties  in  growing 
dysentery  bacilli  and  work  was  therefore  begun  ( 1 )  to  differentiate  the 
true  dysentery  bacilli,  which  are  universally  recognized  as  pathogenic, 
from  the  atypical  or  dysentery-like  organisms  (B.  ambiguus,  B.  alka- 
lescens,  and  B.  dispar)  many  strains  of  which  are  nonpathogenic  and 
whose  etiologic  significance  is  questionable;  (2)  to  devise  a  more 
dependable,  and  if  possible  more  simple  medium,  than  the  nutrient  agar 
(phenolphthalein  titration)  for  the  isolation  of  dysentery  bacilli. 

The  nomenclature  in  the  group  of  dysentery  bacilli  has  become 
quite  confused.  In  this  paper  the  following  will  be  adhered  to: 
B.  dys.  Shiga  corresponds  to  the  original  Shiga-Kruse  mannite  negative 
type.  The  term  B.  flexneri  includes  both  the  B.  dys.  Flexner  and 
Y  types,  and  when  possible  it  will  be  qualified  with  the  race  of  the 
strain,  such  as  V,  W,  X,  Y  or  Z.  The  terms  B.  dys.  Flexner  and 
B.  dys.  Y  are  used  in  their  old  significance. 

Serologic  tests  and  studies  on  classification  were  beyond  the  scope  of  the 
investigation.  Agglutination  with  stock  Flexner  and  Y  serums  were  carried 
out  with  59  cultures.  Acid  production  in  a  number  of  sugars  and  other  fer- 
mentable substances,  as  well  as  the  reactions  in  milk  and  the  indol  test,  were 
observed  on  all  the  stains. 

A  total  of  111  cultures  were  considered  in  this  study.  These  were  dis- 
tributed as  follows:  B.  dys.  Shiga,  17;  B.  ambiguus,  5;  B.  flexneri,  60;  B.  alka- 
lescens,  12;  B.  dispar,  11;  miscellaneous,  6. 

The  Shiga  cultures,  with  one  exception,  were  stock  strains  found  at  the 
Central  Medical  Laboratory  or  the  Army  Medical  School;  several  were 
duplicates. 

The  ambiguus  strains  included  3  (67,  68  and  69)  from  Dr.  Andrews,  St. 
Bartholomew's  Hospital,  London.  One  (4)  was  found  at  the  Central  Medical 
Laboratory  marked  B.  dys.  Shiga  Fletcher  vaccine  stain,  and  another  (101) 
obtained  from  the  Army  Medical  School  and  probably  a  duplicate  of  (4),  was 
marked  B.  dys.  Shiga  Fletcher  1.  Serologic  tests  were  not  made  with  (101). 
The  other  (4)  failed  to  agglutinate  with  several  Shiga  serums,  and  as  both  were 
positive  for  indol  they  are  here  considered  as  B.  ambiguus. 

The  60  cultures  of  B.  flexneri  include  strains  isolated  during  the  war  and 
also  standard  stock  cultures.  Included  in  this  group  are  the  old  Flexner  and 
Y  types  and  authentic  strains  of  the  English  groups  V,  W,  X,  Y,  Z,  VZ  and  WX, 
which  were  sent  me  by  .Dr.  Andrews. 

Received  for  publication  Feb.   16,   1920. 


M.  LEVINE  . 


There  were  12  strains  of  B.  alkalescens  and  11  of  B.  dispar.  These  were 
received  from  Dr.  Andrews  or  freshly  isolated  in  laboratories  of  the  A.  E.  F. 

Of  the  6  miscellaneous  strains  two  (37  and  57)  were  marked  B.  ambiguus. 
They  produced  a  green  fluorescence  in  broth  and  on  gelatin  and  were  so  different 
culturally  from  the  strains  of  B.  ambiguus  received  from  Dr.  Andrews  that  it 
seems  they  should  not  be  considered  as  of  the  same  group.  Two  cultures  (48 
and  108)  were  marked  B.  dys.  Sonne.  They  did  not  agglutinate  with  the  Flexner 
or  Y  serums  available.  Lactose  was  fermented  with  acid  formation  and  then 
became  alkaline.  Milk  was  turned  acid  but  not  coagulated.  These  cultures 
resemble  markedly  some  of  the  B.  dispar  of  Andrews,  at  least  culturally.  One 
strain,  3,  supposedly  a  Shiga,  produced  acid  from  sucrose  and  gave  indol.  It 
was  not  agglutinated  with  a  Shiga  serum.  Another  strain,  97,  differed  from 
all  of  the  other  cultures  studied  in  that  it  fermented  the  glucoside  salicin  with  a 
strong  acidity  in  24  hours. 

AGGLUTINATION  WITH  FLEXNER  AND  Y  SERUMS 

Agglutination  was  made  with  living  24-hour  broth  cultures  of  59  strains. 
The  strains  of  B.  dys.  Shiga,  B.  alkalescens,  and  B.  dispar  were  not  agglutinated 
by  either  of  the  serums.  B.  dys.  Sonne  (48)  and  one  of  the  English  B.  flexneri  Z 
race  (53),  were  also  not  agglutinated.  It  was  noticed  that  the  Z  and  X  races 
of  B.  flexneri  were  only  agglutinated  in  the  low  dilutions,  and  that  (13  and  38) 
the  original  Mt.  Desert  Y  and  the  Oxford  Y  strain,  respectively,  were  not 
agglutinated  even  in  1 :  100  by  the  Y  serum  employed.  From  these  observations 
it  appears  quite  evident  that  what  is  regarded  as  trje  Y  type  of  dysentery  in 
different  laboratories  is  not  of  the  same  serologic  group. 

BIOCHEMICAL  REACTIONS  (TABLE  1) 

All  strains  were  gram-negative  short  rods,  and  nonmotile  as  determined  in 
semisolid  agar  (0.5%  agar  in  broth). 

TABLE     1 

ACID   PRODUCTION   AND   INDOL    (PERCENTAGE  OF   POSITIVE   REACTIONS)    BY   DYSENTERY   AND 

CLOSELY  ALLIED   BACILLI 


Organism 

No.  of 

Strains 

Man- 
nitol 

Lac- 
tose 

Gly- 
cerol* 

Dex- 
trin 

Dul- 
citol 

Su- 
crose 

Xyl- 
lose 

Raffi- 
nose 

Rham- 
nose 

In- 
dol 

B.  dys.  Shiga  

17 

0.0 

0.0 

0.0 

00 

00 

00 

0  0 

0  0 

0  0 

00 

B.  ambiguus  
B.  dys.  flexnerif  
B  alkalescens 

5 
59 
12 

0.0 
100.0 
100  0 

0.0 
0.0 
0  0 

0.0 

0.0* 
1000 

0.0 
40.0 
00 

0.0 
0.0 

1000 

0.0 
64.4 

oo 

0.0 
0.0 
100  0 

0.0 
79.7 
0  0 

100.0 
16.9 
100  0 

100.0 
83.1 
100  0 

B  dispar 

11 

1000 

1000 

81  3 

00 

18  2 

81  8 

81  8 

91  9 

1000 

81  8 

*  Slight  acidity  in  5-7  days  but  more  alkaline  than  PH  7.0. 
t  Includes  all  mannite  fermenting  true  dysentery  bacilli. 

Tests  for  acid  production  were  made  on  glucose,  mannitol,  lactose,  glycerol, 
sucrose,  dextrin,  arabinose,  dulcitol,  rhamnose,  xylose,  raffinose  and  salicin. 
The  medium  employed  consisted  of  1%  peptone,  and  0.4%  dipotassium  phosphate 
with  1%  of  the  test  material.  The  rosolic  acid-china  blue  mixture  of  Bronfen- 
brenner  was  the  indicator.  Incubation  was  at  the  body  temperature,  and  obser- 
vations were  made  daily  for  7  days. 

The  indol  reaction  was  determined  from  peptone  water  after  5  days'  incu- 
bation by  the  nitroso-indol  reaction.  Litmus  milk  was  observed  for  13  days 

Table  1  indicates  that  the  5  main  types  of  dysentery  and  dysentery-like 
organism  may  be  readily  differentiated  by  fermentation  and  indol  reactions 


DYSENTERY  AND  ALLIED  BACILLI  5 

Thus  B.  dys.  Shiga  and  B.  ambiguus  may  be  distinguished  from  the  others 
(B.  flexneri,  B.  alkalescens,  and  B.  dispar)  by  the  inability  of  the  former  to  give 
acid  from  the  alcohol  mannitol.  They  differ  from  each  other  in  that  B.  ambiguus 
forms  indol  and  ferments  rhamnose. 

B.  flexneri  may  be  differentiated  in  a  large  proportion  of  instances  from 
B.  alkalescens  and  B.  dispar  by  the  reaction  in  glycerol  and  xylose.  None  of 
the  Flexner  strains  produced  acid  from  xylose,  whereas  this  substance  was  fer- 
mented vigorously  by  21  of  23  strains  of  B.  alkalescens  and  B.  dispar.  Differ- 
entiation by  glycerol  fermentation  was  not  so  distinct,  as  a  number  of  the 
Flexner  strains  produced  a  small  amount  of  acid.  Quantitative  studies  showed 
that  this  acidity  was  never  beyond  the  true  neutral  point  PH  7.0.  in  5  days. 
With  the  indicator  employed,  however,  the  results  might  be  confusing  in 
inexperienced  hands. 

B.  flexneri  differs  also  from  B.  alkalescens  and  B.  dispar  in  the  milk  reac- 
tion. The  former  produces  a  faint  acidity  in  litmus  milk,  which  reverts  very 
slowly,  if  at  all,  to  a  neutral  reaction  in  from  10-13  days.  B.  alkalescens,  on 
the  other  hand,  reverts  relatively  rapidly,  from  4-8  days,  to  a  distinct  alkaline 
reaction,  while  B.  dispar  becomes  progressively  more  acid,  eventually  coagu- 
lating the  medium.  Unfortunately  the  milk  reaction  has  not  given  concordant 
results  in  the  hands  of  different  observers,  many  recording  distinct  alkalinity 
and  others  coagulating  with  true  dysentery  strains  of  B.  flexneri  type. 

It  remains  to  differentiate  B.  alkalescens  from  B.  dispar.  The  milk  reac- 
tion has  been  referred  to.  The  objectionable  features  of  this  rea'ction  are  the 
variability  of  different  batches  of  milk  and  slowness  of  the  test.  The  lactose 
fermentation  of  B.  dispar,  although  distinct,  is  often  long-delayed.  Table  1 
shows  that  although  there  is  some  overlapping,  the  two  organisms  are  markedly 
different  when  groups  of  characters  rather  than  single  reactions  are  considerel. 
Thus  B.  alkalescens  does  not  form  acid  from  lactose,  sucrose  or  raffinose, 
but  attacks  dulcitol  vigorously,  while  B.  dispar  rarely  ferments  dulcitol,  but 
does  form  acid  from  lactose  and  most  always  from  sucrose  (81.8%)  and 
raffinose  (91.9%).  B.  alkalescens  seems  to  be  a  very  homogenous  group. 
B.  dispar  probably  consists  of  several  varieties.  The  indol-negative,  'xylose- 
negative  variety  of  B.  dispar  corresponds  culturally  to  the  strain  isolated  by 
Sonne  in  Denmark. 

VARIETIES    OF   B.    FLEXNERI 

A  number  of  subdivisions  of  the  mannite  fermenting  dysentery  strains  on 
serologic  and  biochemical  reactions  have  been  proposed  in  the  past.  The 
probable  untenability  of  B.  dys.  Y  as  distinguished  from  B.  dys.  Flexner  has 
already  been  referred  to.  The  differentiation  of  B.  flexneri  by  the  English 
War  Committee  as  determined  by  careful  absorption  tests  into  V,  W,  X,  Y 
and  Z  races  appears  much  more  acceptable  and  desirable. 

The  value  of  differentiation  of  this  group  on  fermentation  reactions  has 
fallen  into  disrepute  of  late.  Thus  the  fermentation  of  maltose,  sucrose  and 
dextrin,  which  were  formerly  emphasized  as  differentiating  varieties  of  mannite 
fermenting  dysentery  strains,  is  about  to  be  discarded.  Maltose  was  not 
employed  in  this  study  as  it  was  considered  unreliable  on  account  of  the 
difficulty  in  obtaining  a  product  entirely  free  from  glucose,  and  the  ease  with 
which  it  decomposes  on  sterilization.  Of  the  tests  tried  with  59  strains  of 
B.  Flexneri  the  following  positive  results  were  obtained  with  substances  that 
might  be  of  value  for  subdivision :  sucrose,  64.4% ;  dextrin,  40.% ;  rhamnose, 
16.9%;  raffinose,  79.7%;  and  indol,  83.1%.  The  correlation  coefficients  for  each 


M.  LEVINE 


pair  of  characters  is  given  in  table  21  which  shows  rhamnose  correlates  best 
with  the  other  characters.  Subdividing  on  rhamnose,  two  groups  are  obtained 
as  follows : 


Rhamnose  + 
Rhamnose  — 


Strains 
10 


Sucrose 
80 
60 


-Percent     Positive 


Dextrin 
70 


Raffinose 

0 

94 


Indol 
100 


TABLE     2 
CORRELATION    COEFFICIENTS    FOR    FERMENTATIVE    CHARACTERS 


Sucrose 

Dextrin 

Rhamnose 

1 
Raffinose    ! 

Indol 

Sucrose 

+0  62 

+0  43 

006 

+0  35 

Dextrin.              

+062 

+  065 

—  0  46 

+0  50 

Rhamnose  

+0.43 

+0.65 

—  1  00 

+1  00 

Raffinose                         .  . 

—006 

—  0  46 

—  1  00 

0  45 

Indol      ..           .                .... 

+0.35 

+0.50 

+1.00 

—  0  45 

i 

Raffinose  fermentation  is  particularly  interesting.  Of  30  strains  in  the 
rhamnose-negative  subgroup  which  fermented  sucrose,  all  attacked  raffinose ; 
but,  of  8  sucrose  fermenters  in  the  rhamnose-positive  subgroup  none  attacked 
the  trisaccharid.  The  source  of  the  10  rhamnose  fermenting  strains  was: 
Strain  26  was  isolated  at  the  Cent.  Med.  Dept.  Lab.  from  a  patient  and  diag- 
nosed as  probably  B.  dys.  Y;  (60  and  61)  were  isolated  at  Lab.  1,  A.  E.  R, 
from  a  patient  and  carrier,  respectively,  and  reported  as  B.  dys.  Flexner  and 
B.  dys.  Y.  and  sent  in  for  further  identification.  As  the  foregoing  diagnoses 
were  based  merely  on  the  two  serums  available — Flexner  and  Y — the  designa- 
tions should  not  be  accepted  as  final.  It  would  be  desirable  to  know  to  which 
race  of  Flexner  bacilli  they  belong.  The  remaining  7  strains  were  received 
from  Dr.  Andrews  and  Dr.  Inman  of  London.  One,  94,  was  labelled  B. 
flexneri  Y  race  which  is  a  sort  of  composite  of  the  V,  W,  X  and  Z  races. 
The  other  6  strains  were  all  B.  flexneri  Z  race.  Thus  there  seems  to  be  a 
correlation  between  rhamnose  fermentation  and  the  Z  race  of  B.  flexneri. 
If  subdivision  is  to  be  made  at  all  on  fermentation  reactions,  then  it  appears 
that  rhamnose  would  be  the  logical  choice. 

ACID   PRODUCTION   FROM    GLUCOSE 

In  order  to  devise  a  medium  for  the  differentiation  of  B.  alkalescens  and 
B.  dispar  from  the  other  dysentery  or  dysentery-like  strains,  the  effects  of 
various  constituents  of  a  selected  medium  on  the  rate  of  acid  production  and 
reversion  were  studied.  Ten  cultures,  2  B.  dys.  Shiga,  2  B.  dys.  Y,  2  B.  dys. 
Flexner,  2  B.  alkalescens,  and  2  B.  dispar  were  chosen  for  study. 

Concentration  of  glucose. — The  medium  consisted  merely  of  peptone  (Difco) 
1%  dipotassium  phosphate  0.4%,  and  glucose  in  varying  amounts  0.0  to  0.5%, 
prepared  in  the  following  manner:  To  1,000  cc  of  distilled  water  in  a  flask 
was  added  10  gm.  of  peptone,  4  gm.  of  dipotassium  phosphate,  and  the  flask 
was  then  heated  until  the  contents  were  dissolved  (about  20-30  minutes). 
The  medium  was  then  filtered  through  paper  and  enough  of  a  freshly  pre- 
pared 10%  glucose  solution  was  added  to  give  the  desired  concentration  of 
the  carbohydrate.  The  medium  was  tubed  (about  20-25  c  c)  and  sterilized  in 
the  autoclave  10  minutes  at  10  pounds,  after  which  it  was  incubated  to  elim- 
inate unsterile  tubes. 


1  See  Levine,  Jour.  Infect.  Dis.,   1918,  3,  p.  253. 


DYSENTERY  AND  ALLIED  BACILLI 


Inoculation  was  made  with  0.1  cc  of  a  24-hour  broth  culture  with  incuba- 
tion at  body  temperature. 

H-ion  concentration  was  determined  daily  for  4  days  by  withdrawing  Ice 
of  the  culture  into  4  c  c  of  neutral  distilled  water  (Pn  7.0)  in  a  clean,  flat 
bottomed  test  tube,  and  after  adding  the  required  amount  of  an  appropriate 
indicator,  the  color  was  matched  with  H-ion  standards.  Great  difficulty  was 
encountered  in  obtaining  neutral  distilled  water  in  the  laboratory  in  France. 
It  was  found,  however,  that  the  error  introduced  by  the  neutralization  of 
ordinary  distilled  water  with  a  small  amount  of  sodium  hydroxid  was  only 
about  0.1,  which  was  within  the  limits  of  error  in  reading.  Such  neutralized 
water  had  to  be  freshly  prepared  and  quickly  utilized. 

&/JYS. 


I 

\ 

I 


Chart  1. — Effects  of  peptone,  dipotassium  phosphate  and  glucose  on  the  rate  of  acid  pro- 
duction and  reversion. 

It  was  concluded  (1)  that  with  1.0%  peptone  and  0.4%  dipotassium  phos- 
phate, the  employment  of  0.3%  or  more  glucose  was  undesirable  for  the  purpose 
of  differentiating  B.  alkalescens  and  B.  dispar  from  the  true  dysentery  bacilli ; 
(2)  that  in  the  absence  of  glucose  B.  alkalescens  and  B.  dispar  form  alkali 
more  rapidly  than  the  true  dysentery  strains  (Shiga  and  B.  flexneri)  ;  (3)  that 
with  0.1%  glucose  there  is  reversion  among  the  true  dysentery  strains,  but 
B.  alkalescens  and  B.  dispar  revert  much  more  rapidly;  (4)  that  with  0.2% 
glucose  reversion  among  the  true  dysentery  cultures  was  greatly  inhibited, 
whereas  B.  alkalescens  and  B.  dispar  showed  a  marked  alkali  production  after 
the  primary  acidity,  as  is  shown  in  chart  1. 


8  M.  LEVINE 

Concentration  of  Peptone. — The  following  experiment  was  made  in  Wash- 
ington to  determine  the  effect  of  the  concentration  of  peptone  on  acid  produc- 
tion and  reversion: 

Three  batches  of  medium  (0.2%  glucose,  0.4%  dipotassium  phosphate,  and 
1.0%,  1.5%  and  2.0%  peptone  respectively)  were  prepared  as  described;  7  cc 
portions  were  placed  in  tubes,  autoclaved  at  10  pounds  for  10  minutes  and 
incubated  for  48  hours  to  eliminate  unsterile  tubes. 

Seven  tubes  of  each  medium  were  inoculated  from  24-hour  cultures  of 
organisms  and  incubated  at  37  C. 

Acidity  determinations  were  made  by  the  comparator  in  place  of  the  dilu- 
tion method  previously  described.  Two  duplicate  cultures  were  taken  and  to 
one  was  added  0.3  c  c  of  an  appropriate  indicator  and  the  color  matched  with 
standards,  the  duplicate  tube  being  employed  to  correct  the  error  due  to  the 
color  and  turbidity  of  the  culture  medium  in  the  comparator  test.  This  tube 
was  reincubatel  and  employed  for  this  purpose  in  acidity  determinations  on 
subsequent  days. 

The  concentration  of  peptone  did  not  influence  acidity  production  nor  rever- 
sion of  the  true  dysentery  bacilli  nor  of  B.  ambiguus,  but  that  with  B.  alkalescens 
and  B.  dispar  reversion  was  much  more  rapid  with  1.5%  peptone  than  when 
1.0%  peptone  was  employed.  Increasing  the  concentration  to  2.0%  did  not 
further  increase  the  rate  of  reversion. 

Comparing  the  results  with  1.0%  peptone  with  those  previously  obtained  in 
the  original  experiment  in  France,  reversion  was  somewhat  delayed  in  the 
new  series.  Although  an  adequate  explanation  is  not  available,  it  is  felt  that 
the  difference  is  probably  due  to  a  difference  in  the  actual  concentration  of 
glucose.  The  glucose  available  overseas  was  probably  not  thoroughly  anhydrous. 

Aeration  seems  to  increase  the  rate  of  alkali  production,  after  the  primary 
acidity,  in  the  case  of  B.  alkalescens  and  B.  dispar. 

To  determine  whether  the  differentiation  indicated  in  the  quantitative  obser- 
vations could  be  applied  qualitatively,  each  organism  was  inoculated  in  duplicate 
into  the  peptone  phosphate  medium  containing  as  indicators  1%  of  a  0.5% 
phenol-red  and  1%  of  a  0.2%  brom-cresol-purple,  respectively,  and  incubated 
at  37  C.  Records  of  acidity  were  made  daily. 

With  exception  of  (37  and  57),  which  have  been  referred  to  as  probably  mis- 
placed in  this  group,  and  which  remained  alkaline  throughout  the  experiment, 
all  other  cultures  were  distinctly  acid  to  both  indicators  after  24  hours'  incuba- 
tion. With  brom-cresol-purple  as  the  indicator,  all  cultures  of  B.  alkalescens  and 
B.  dispar  showed  reversion  to  distinct  purple-blue  color,  as  did  also  one  of  the 
B.  dys.  Sonne  after  3  days'  incubation.  The  true  dysentery  strains  and  B. 
ambiguus  were  yellow  or  brownish  in  color.  On  further  incubation  (6  days), 
the  other  strain  of  B.  dys.  Sonne  and  one  B.  flexneri  became  distinctly  alkaline 
and  a  number  of  the  true  dysentery  cultures  began  to  show  some  reversion, 
thus  obscuring,  though  not  eliminating,  the  differentiation. 

With  phenol-red,  on  the  other  hand,  all  cultures  of  B.  dys.  Shiga  and  B. 
ambiguus,  and  all  but  one  of  B.  flexneri  were  distinctly  acid  for  6  days.  The 
12  B.  alkalescens  strains  were  distinctly  alkaline.  Two  of  the  11  B.  dispar  were 
neutral,  the  others  distinctly  alkaline.  One  B.  dys.  Sonne  was  neutral  and 
another  alkaline. 

Rate  of  Acid  Production. — It  was  observed  that  glucose  was  attacked  more 
rapidly  by  B.  alkalescens  and  B.  dispar  than  by  the  other  organisms  of  this 
collection.  Inoculation  was  from  24-hour  broth  cultures  (0.1  cc  to  30  cc  of 


DYSENTERY  AND  ALLIED  BACILLI  9 

medium)    and   H-ion   determinations   were  made  by  the   dilution  method.     In 
chart  2  the  data  are  shown  graphically. 

The  rate  of  acid  production  was  observed  qualitatively  by  the  use  of  brom- 
cresol-purple  and  in  some  instances  with  the  china-blue  rosolic  acid  indicator. 
Inoculation  was  made  from  24-hour  agar  slants;  incubation  was  at  37  C.  in 
the  ordinary  manner;  acidity  was  recorded  after  6  hours.  At  this  time  all 
strains  of  B.  alkalescens  and  B.  dispar,  one  B.  flexneri  and  the  2  B.  dys. 
Sonne  were  distinctly  acid  as  indicated  by.  a  distinct  or  dirty  yellow  with  brom- 
cresol-purple.  All  other  strains  produced  acid  less  rapidly.  They  showed  a 
distinct  purple  (more  alkaline  than  PH  6.3)  with  brom-cresol-purple  and  with 
the  china-blue  mixture  were  colorless  or  light  blue. 


Q? 

i 


S.O 


6.0 


Chart  2. — Rate  of  acid  production:  Inoculations  made  from  24-hour  broth  cultures  and 
H-ion  determinations  made  by  dilution  method. 

It  may  be  concluded  from  the  observations  of  glucose  fermentation  that  B. 
alkalescens  and  B.  dispar  produce  acid  more  rapidly  and  then  revert  to  a 
distinctly  alkaline  reaction  that  may  be  indicated  qualitatively,  by  phenol-red 
or  brom-cresol-purple.  The  use  of  brom-cresol-purple,  however,  would  require 
experience  and  care,  whereas  phenol-red  necessitates  a  prolonged  period  of  incu- 
bation. The  most  desirable  indicator  for  qualitative  differentiation  would  be  one 
which  changes  at  PH  6.5  showing  a  distinct  coloration  on  the  alkaline  side. 


A   SIMPLIFIED    SOLID    MEDIUM    FOR   GROWTH    AND   ISOLATION   OF 
DYSENTERY    BACILLI 

After  a  number  of  preliminary  experiments  it  was  found  that  by  the  addi- 
tion of  a  small  amount  of  glucose  (0.03-0.05)  to  peptone-phosphate  agar, 
growths  as  luxuriant,  if  not  more  so,  than  on  nutrient  agar  could  be  obtained. 
As  facilities  for  determination  of  the  optimum  H-ion  concentration  were  not 
available  at  the  time  in  France  a  series  of  experiments  were  carried  out  to 
determine  the  optimum  concentration  of  dipotassium  phosphate  for  growth  of 
dysentery  bacilli  in  a  medium  not  requiring  any  further  adjustment  of  reaction. 


10  M.  LEVINE 

The  medium  consisted  of  1.0%  peptone,  0.1%  glucose  and  0.2  to  0.7%  phos- 
phate. Inoculation  of  the  agar  plates  was  made  from  a  24-hour  broth  culture. 
A  concentration  of  0.4-0.5%  of  the  phosphate  gave  best  results  with  6  cultures 
examined.  It  is  interesting  in  this  connection  to  note  that  the  titratable  acidity 
with  phenolphthalein  was  in  each  instance  -f  0.7%.  The  H-ion  concentration 
was  much  more  varied,  probably  7.1  with  the  0.2%  of  the  phosphate  and  7.8 
with  the  0.7%  of  the  buffer  salt,  as  indicated  by  subsequent  experiments. 

The  influence  of  the  H-ion  concentration  on  the  growth  of  the  dysentery 
bacilli  seems  marked  on  solid  medium.  It  has  been  my  experience  that  in 
liquid  medium  the  effect  of  the  H-ion  concentration  is  not  so  evident. 

Experiments  on  the  effect  of  the  concentration  of  dipotassium  phosphate 
repeated  with  39  strains  using  3  concentrations  of  the  salt  (0.2,  0.45  *and  0.7%, 
respectively),  showed  that: 

1.  The  phenolphthalein  titration  is  a  poor  index  of  the  true  acidity  of  the 
medium.    The  variation  in  the  titrable  acidity  was  close  to  the  limit  of  experi- 
mental error,  whereas  the  difference  in  H-ion  concentration  with  the  different 
quantities  of  phosphate  was  marked  and  distinct. 

2.  The  optimum  reaction  is  not  the  same  for  all  strains  of  dysentery.    Two 
(5.1%)   grew  best  with  the  largest  quantity  of  phosphate,  four   (10.2%)    with 
the  least  amount  of  phosphate  and  twelve  (30.7%)  show  their  optimum  growth 
when  0.45%  of  dipotassium  phosphate  was  used.    Seventeen  (43.6%)  did  equally 
well  on  all  of  the  3  mediums.     Considering  all  cultures,  we  find  33  (84.7%)  to 
have  done  as  well  or  better  with  0.45%  phosphate  than  on  either  of  the  other 
concentrations  of  this  salt.     The  H-ion  concentration  with  0.4%  of  the  phos- 
phate is  generally  7.4  or  7.5.    This  quantity  was  selected  as  probably  the  most 
reliable  and  desirable. 

Choice  of  Indicator. — A  distinct  and  noninhibitory  indicator  is  an  important 
adjunct  to  the  successful  isolation  of  dysentery  bacilli.  It  was  hoped  that  the 
eosin  and  methylene-blue  combination  of  Holt,  Harris  and  Teague,  which  was 
found  so  valuable  in  water  work,  might  be  successfully  employed,  particularly 
as  it  was  reputed  to  be  noninhibitory.  Thirty-nine  strains  of  dysentery  bacilli 
were  inoculated  on  agar  with  and  without  the  indicator  from  a  24-hour  peptone 
phosphate  culture. 

The  composition  of  the  medium  was: 

Agar  1.5% 

Peptone   1.0% 

Dipotassium  phosphate 0.4% 

Glucose 0.1% 

Indicator  per  100  c  c  of  above 

Eosin  2%  yellowish  aq 2.0  c  c 

Methylene-blue  0.5%  aq .' 2.0  c  c 

(The  PH  of  this  medium  was  7.5). 

B.  dys.  Shiga  was  markedly  inhibited.  A  slight  growth  was  observed  on 
prolonged  incubation  (48-72  hours).  Sixteen,  or  50%,  of  the  mannite  fermenting 
dysentery  strain  were  partially  inhibited. 

Of  a  number  of  indicators  tried,  the  china-blue  rosolic  acid  mixture  was 
found  to  be  the  least  inhibitory  when  working  with  pure  cultures.  Similar 
results  were  obtained  with  artificial  suspensions  of  dysentery  organisms  in 
normal  stools. 


DYSENTERY  AND  ALLIED  BACILLI  11 

SUMMARY   AND    CONCLUSIONS 

Observations  made  on  111  strains  of  dysentery  and  dysentery-like 
organisms  indicate: 

1.  The  strains  of  B.  dysenteriae  Y  used  in  different  laboratories  are 
not  of  the  same  serologic  group. 

2.  The  main  groups  of  the  dysentery  and  closely  allied  bacilli, 
B.  dys.  Shiga,  B.  flexneri,  B.  ambiguus,  B.  alkalescens  and  B.  dispar, 
are  readily  differentiated  by   fermentation  reactions.     B.  dys.   Sonne 
appears  to  be  intermediate  between  B.  dispar  and  B.  flexneri. 

3.  Subdivision  of  B.  flexneri  on  fermentation  reactions  is  not  advis- 
able, but  the  flexneri  Z  race  seems  to  be  characterized  by  acid  pro- 
duction from  rhamnose.     This  character  is  also  strikingly  correlated 
with  an  inability  to  attack  raffinose  when  sucrose  is  fermented. 

4.  B.  alkalescens  and  B.  dispar  form  acid  from  glucose  rapidly  in 
a  medium  containing  1.5%  peptone,  0.4%  dipotassium  phosphate,  and 
0.2%  glucose,  then  revert  rapidly  to  an  alkaline  reaction.     B.  dys. 
Shiga,  B.  flexneri  and  B.  ambiguus  form  acid  less  rapidly  -and  remain 
permanently  acid  or  revert  slowly. 

5.  Dyes,   such  as   eosin  and   methylene-blue,   the   fuchsin-sulphite 
indicator,  and  excess  of  rosolic  acid  or  china-blue  were  found  to  inhibit 
many  strains  of  dysentery,  particularly  the  Shiga  type. 

6.  The  following  medium  is  suggested  for  isolation  work : 

Distilled  water  1,000  c  c 

Agar  15  gm. 

Peptone   10  gm. 

Dipotassium  phosphate 4  gm. 

To  each  100  c  c  of  the  melted  medium  add  before  using: 

Lactose,  20%  solution 5.0  c  c 

Glucose,  5%  solution 1.0  c  c 

Rosolic  acid  (1.0%  in  90%  alcohol) '. .  1.0  c  c 

China-blue  (0.5%  in  water) 1.0  c  c 

The  H-ion  concentration  of  this  medium,  which  requires  no  adjust- 
ment of  reaction  and  does  not  need  to  be  filtered  when  used  on  plates, 
is  7.4  to  7.5. 


With  the  compliments  of  the  author 


Reprinted  from  THE  JOURNAL  OF  THE  AMERICAN  WATER  WORKS  ASSOCIATION 
Vol.  9,  No.  2,  March,  1922 


A   FACULTATIVE   SPORE-FORMING   LACTOSE-FERMENT- 
ING ORGANISM  FROM  IOWA  SURFACE  WATERS, 
(B.  MACERANS)1 

BY  JACK  J.  HINMAN,  Jn.,2  AND  MAX  LEVINES 

The  occasional  presence  of  sporing  lactose-fermenters  in  water 
capable  of  growing  aerobically  has  been  reported  by  Meyer,  Ewing, 
Ellms,  Perry  and  Monfort,  and  Hall  and  Ellefson,  but  very  little 
is  known  as  to  the  source  or  biology  of  these  forms. 

In  the  course  of  routine  water  analyses  at  the  Iowa  State  Water 
Laboratory  it  appeared  that,  with  chlorinated  surface  waters  the 
proportion  of  unconfirmed  presumptive  tests  was  excessive,  when 
using  preliminary  enrichment  in  lactose  broth,  and  eosin  methylene 
blue  agar  for  confirmation.  With  litmus  lactose  agar,  however, 
atypical  colonies  were  not  infrequently  obtained,  which  often  formed 
gas  when  fished  to  lactose  broth  and  which  would  therefore  be  re- 
garded as  members  of  the  colon  group,  on  the  basis  of  the  Treasury 
Department  Standard.  These  organisms  were  invariably  negative 
for  gas  formation  in  lactose  bile.  It  occurred  to  us  that  possibly 
aerobic  sporing  lactose  bacilli  might  be  responsible,  for  a  part  at 
least,  of  these  atypical  reactions,  and  attempts  were  made  to  isolate 
them. 

Table  1  has  been  prepared  to  show  the  frequency  with  which 
presumptive  tests  upon  filtered,  chlorinated  Iowa  city  waters  were 
found  to  be  organisms  which  could  not  be  confirmed  as  B.  coli  or 
B.  aerogenes.  It  appears  that  those  waters  which  were  taken  from 
the  Iowa  River,  or  from  the  Mississippi  River  below  the  junction  of 
the  Iowa  with  the  Mississippi,  are  particularly  likely  to  contain 
these  non-confirmed  organisms.  This  apparent  peculiarity  may  be 
due,  however,  to  the  greater  number  of  samples  examined  from  the 
cities  of  Burlington,  Iowa  City  and  Keokuk  where  such  water  is 
handled. 

1  Presented  before  the  Iowa  Section  meeting,  November  1, 1921. 
*  Associate  Professor  of  Sanitation,  State  University  of  Iowa. 
» Department  of  Bacteriology  and  Pathology,  Iowa  State  College. 

330 


SPORE-FORMING   ORGANISM   FROM  IOWA 


331 


From  two  sources,  Iowa  City  and  Burlington,  Iowa,  14  pure  cul- 
tures have  been  isolated  and  studied  as  to  their  morphological, 
cultural  and  other  characteristics.  Considerable  difficulty  was  en- 
countered in  effecting  separation  from  non-fermenting,  sporing 

TABLE  1 
Fermentation  tests  on  treated  Iowa  city  waters,  March  3, 1914,  t°  December  SO, 1920 


CITY 

1  CC.  WATEK 

10  CC.  WATEE 

1 

ffl 

1 

1 

« 

Positive  presump- 
tive tests  not 
confirming 

a 

| 

1 

1 

1 

PQ 

8 

|l 
|P 

®^§ 

in 

No  gas  formation 

Burlington 

22 
0 
9 
2 
0 
3 
2 
1 
3 
35 
11 
3 
0 
0 
3 

2 
1 
1 
0 
0 
2 
0 
0 
1 
2 
0 
1 
0 
0 
0 

41 
7 
14 
2 
2 
5 
2 
5 
2 
312 
22 
2 
1 
4 
0 

651 
11 
54 
11 
4 
27 
25 
8 
11 
3868 
101 
2 
7 
8 
16 

71 
3 
33 
12 
0 
11 
12 
1 
14 
62 
56 
4 
0 
0 
18 

22 
4 
5 
1 
0 
11 
0 
0 
7 
6 
8 
8 
0 
0 
3 

584 
21 
40 
12 
8 
12 
28 
2 
10 
492 
258 
2 
5 
5 
2 

1852 
23 
37 
12 
8 
34 
80 
1 
17 
1147 
135 
0 
7 
10 
21 

Cedar  Rapids  

Centerville    

Chariton 

Council  Bluffs  

Creston  

Davenport           

Fairfield  

Ft.  Madison  

Iowa  City  

Keokuk 

Lenox 

Oskaloosa  

Ottumwa  

Storm  Lake 

94 

10 

421 

4804 

297 

75 

1481 

3384 

Total  number  of  fermentation  tubes 10,566 

Total  number  positive 2,378 

Total  number  positive,  non-confirming 1,902 

Per  cent  positive  tubes  non-confirming 80  per  cent 

Per  cent  all  tubes  non-confirming  positive 18  per  cent 

Burlington,  Iowa  City,  Keokuk: 

Per  cent  positive  tubes  non-confirming 85 .3  per  cent 

Per  cent  all  tubes  non-confirming  positive 17  per  cent 

aerobes,  which  seemed  to  grow  associatively.  The  method  which 
proved  most  successful  was  (1)  to  grow  in  lactose  (Andrade)  broth, 
(2)  to  plate  on  lactose  (Andrade)  agar  as  soon  as  acid  developed,  (3) 
fish  acid  colonies  to  lactose  (Andrade)  agar  slants  (inoculate  sur- 


332  JACK   J.    HINMAN,  JR.,    AND   MAX   LEVINE 

face  and  butt)  and  incubate  the  latter  for  72  hours  observing  daily. 
If  pure,  a  transparent  growth  with  acid  on  slant  and  acid  and  gas  in 
butt  will  be  observed.  If  non-fermenting  spore-formers  are  present 
the  surface  growth  becomes  opaque.  Microscopic  examination  of 
Gram's  stain  of  48  to  72  hour  culture  on  lactose  (Andrade)  agar 
should  show  no  spores.  If  spores  were  present,  then  the  purifica- 
tion process  outlined  above  was  repeated,  for  it  was  observed  that 
whenever  this  was  the  case,  a  non-fermenting  aerobic  spore  former 
could  be  isolated  while  the  pure  fermenting  type  did  not  show  spores 
on  this  medium  in  the  designated  time .  The  strains  isolated  resemble 
B.  macerans  described  by  Schardinger. 

MORPHOLOGY 

Vegetative  cells.  The  organism  varies  in  size  on  different  culture 
media. 

On  nutrient  agar,  the  vegetative  cells  appear  (after  24  hours  at  37°C.)  as 
rods  about  as  wide  as  B.  coli,  but  2  to  4  times  as  long.  The  size  of  the  majority 
being  0.6  by  2.5  /x.  They  are  grouped  singly  or  in  pairs;  parallel  forms  were 
frequently  observed  and  occasionally  V  forms  were  noted.  The  ends  are 
rounded  and  the  bacillus  is  slightly  fusiform. 

On  lactose  (Andrade)  agar  and  in  litmus  milk  the  cells  are  somewhat  longer, 
usually  3 1 o  4  n .  Spores  were  not  seen . 

In  lactose  (Andrade)  broth  they  appeared  especially  elongated  often 
measuring  6  and  occasionally  8  AC.  All  of  the  14  cultures  showed  an  occasional 
spore  on  nutrient  agar  after  24  hours  and  after  48  hours  at  37°,  sporangia  and 
spores  were  numerous.  The  endospores  are  elliptical,  their  diameter  greater 
than  that  of  the  vegetative  cells  and  located  sub  terminally.  The  size  of  the 
majority  of  spores  was  0.8  by  1 .4 /*. 

STAINING    REACTIONS 

After  repeated  observations  and  comparison  with  known  cultures 
the  organisms  were  considered  to  be  Gram  negative.  This  was 
particularly  true  if  the  cultures  were  grown  on  lactose  media.  The 
gentian  violet  stain  is  removed  with  difficulty  and  the  Gram  stain 
may  easily  be  confused.  The  technique  employed  was  to  stain  for 
1J  minutes  with  aniline  oil  gentian  violet  then  with  Gram's  iodine, 
to  decolorize  for  5  minutes  with  fresh  95  per  cent  alcohol  and  to 
counter-stain  with  dilute  saffranin. 

The  Gram  stain  showed  the  vegetative  cells  to  exhibit  a  tendency 
to  granulation  but  in  cultures  from  agar  and  Loeffler's  blood  serum 


SPOB  E-FORMING   ORGANISM   FROM   IOWA  333 

no  granules  were  discernible  when  stained  with  carbol  fuchsin, 
Loeffler's  methylene  blue  or  Albert's  diphtheria  stain.  The  vege- 
tative cells  stain  readily  with  all  of  the  stains  mentioned. 

MOTILITY 

All  of  the  14  cultures  were  found  to  be  motile  when  examined  in  a 
hanging  drop  from  a  16  hour,  37°C.  peptone  water  culture. 

SPORE    FORMATION 

It  has  already  been  mentioned  that  spores  were  formed  readily 
on  nutrient  agar  in  2  days  at  the  body  temperature;  it  is  significant 
to  note,  however,  that  spores  could  not  be  demonstrated  in  lactose 
agar  or  milk  even  after  long  incubation. 

Three  cultures  were  inoculated  into  lactose  broth,  lactose  agar 
and  plain  agar  and  incubated  at  body  temperature  for  48  hours. 
No  spores  were  visible  in  the  lactose  broth  or  lactose  agar  but  they 
were  numerous  on  plain  agar. 

On  another  occasion  six  cultures  were  inoculated  into  two  batches 
of  litmus  milk.  Microscopic  examination  (Gram  stain)  after  4  days 
and  again  after  10  days  at  37°C.  did  not  show  any  spores  although 
the  organism  from  these  media  was  found  to  survive  a  temperature 
of  92°C.  for  20  minutes.  There  was  a  marked  tendency  to  granular 
staining. 

Examination  of  four  cultures  on  lactose  (Andrade)  agar,  incubated 
3  days  at  37°C.  then  stored  for  2  weeks  in  the  ice  box  also  failed  to 
disclose  any  spores  when  stained  by  Gram's  method.  As  in  the 
milk  cultures  there  was  a  marked  tendency  to  granular  staining 
and  the  cells  were  resistant  to  heat,  surviving  92°  for  20  minutes. 

We  feel  therefore  that  the  organism  does  not  form  recognizable 
spores  readily,  if  at  all,  on  lactose  media.  This  is  of  some  practical 
significance  in  water  work  as  it  indicates  the  improbability  of  de- 
tecting spores  of  this  bacillus  and  thereby  differentiating  it  from 
B.  coli  by  examination  of  stained  mounts  from  confirmatory  lactose 
agar  plates. 

TEMPERATURE    RELATIONSHIPS 

Growth  is  much  more  luxuriant  on  agar  at  37°C.  than  at  20°  to 
22°C.  At  the  lower  temperature  4  days  may  be  necessary  before 
any  growth  is  visible.  No  growth  was  visible  after  one  week  at 
53°C. 


334  JACK  J.  HINMAN,  JR.,  AND  MAX  LEVINE 

CULTURAL  CHARACTERS 

Plain  agar.  On  this  substrate  the  character  of  growth  varies  with 
the  age  and  consistency  of  the  medium.  On  moist,  fresh  agar,  there 
is  a  moderate,  spreading,  effuse,  glistening,  transparent,  butyrous 
growth  which  is  difficult  to  see  if  the  medium  is  not  perfectly  clear. 
On  dry  (high  agar  content)  or  old  agar  the  growth  is  filiform  and 
almost  opaque  and  in  old  cultures  (2  weeks)  it  becomes  membranous. 
The  medium  is  not  changed,  there  is  no  distinctive  odor  and  no 
chromogenesis. 

Lactose  agar.  On  fresh  lactose  (Andrade)  agar,  the  surface  growth 
is  almost  invisible  on  account  of  its  effuse  character  and  transpar- 
ency. It  spreads  rapidly  over  the  surface  forming  acid,  and  acid 
and  gas  in  butt. 

Gelatin.    At  37°C.  gelatin  was  not  liquefied  in  48  hours. 

At  20°  to  22°C.  gelative  stabs  showed  a  moderate  growth  which 
was  best  at  the  top  and  filiform  along  the  line  of  puncture.  Lique- 
faction was  very  slow,  first  becoming  evident  in  from  14  to  20  days. 

Tubes  evenly  inoculated  on  the  surface  and  kept  for  30  days 
(20°  to  22°C.)  showed  but  2  mm.  of  liquefaction. 

Broth.  In  nutrient  broth  at  37°C.  there  was  but  slight  clouding 
and  very  little  sediment.  Surface  growth  was  not  evident  until 
the  third  day  when  a  pellicle  was  present.  In  sugar  broth  (glucose, 
lactose,  sucrose,  maltose  and  inulin)  there  was  no  surface  growth 
even  on  long  incubation.  (14  days) 

Potato.  The  reaction  on  potato  was  particularly  striking.  In 
24-48  hours  at  body  temperature,  the  entire  mass  of  culture  medium 
was  covered  with  gas  and  in  4  to  7  days,  the  potato  was  almost  com- 
pletely digested.  The  diastatic  action  was  very  marked  with  all  of 
the  14  cultures  studied.  The  organism  evidently  produces  a  power- 
ful pectinase,  as  the  medium  is  entirely  disintegrated. 

COLONY  CHARACTERISTICS 

Plain  agar.  On  nutrient  agar  surface  colonies  when  well  isolated, 
were  irregular  in  form  with  a  lobate  edge.  They  may  be  described 
as  amoeboid.  -  The  colonies  were  smooth,  glossy  and  effuse,  showed 
no  distinct  internal  structure  and  quickly  confluesced.  It  is  gener- 
ally difficult  to  discern  the  colonies  due  to  the  transparency  of  the 
growth. 


SPORE-FORMING   ORGANISM   FROM  IOWA  335 

Subsurface  colonies  resembled  those  of  B.  coli,  i.e.,  they  were 
circular  or  elliptical  with  an  entire  edge  and  showed  a  granular 
internal  structure. 

Litmus  lactose  agar.  The  surface  colonies  in  24  hours  at  37°C. 
were  faintly  acid,  otherwise  resembling  those  described  for  plain 
agar. 

The  subsurface  colonies  were  slightly  acid  resembling  B.  coli. 

Incubation  for  48  hours  increased  the  acid  reaction. 

Endo  agar.  The  medium  employed  was  the  product  supplied  by 
the  Digestive  Ferments  Company.  On  this  medium  B.  coli  was 
observed  to  give  a  distinct  red  colony  but  only  a  slight  metallic 
sheen,  if  any.  Inoculation  was  made  only  on  the  surface. 

The  organism  under  consideration  showed  faint,  but  hardly  dis- 
cernible, growth  in  24  hours  at  37°C.  After  48  hours  small  flat, 
round  and  amoeboid  colonies  about  1  mm.  in  diameter,  pink  to  red, 
with  a  more  intensely  colored  edge  and  center,  were  developed. 
We  would  certainly  not  consider  it  colon-like  but  these  colonies 
would  have  to  be  fished  in  compliance  with  the  United  States  Treas- 
ury Department  Standard. 

Eosin  methykne  blue  agar.  Two  media  were  employed;  the  simpli- 
fied E.  M.  B.  of  Levine  and  the  Difco  product.  The  results  were 
similar.  There  was  no  growth  in  24  hours  and  after  48  hours  small 
pinhead,  discrete  colonies  about  \  mm.  in  diameter  with  a  distinct 
metallic  sheen  were  present. 

GROWTH    IN    LITMUS    MILK 

All  cultures  were  inoculated  into  two  different  batches  of  litmus 
milk,  made  from  skim  milk  powder,  and  incubated  at  37°C. 

The  litmus  was  rapidly  decolorized  (24  hours)  and  the  medium 
acidified.  After  4  days,  coagulation  could  be  induced  by  heating. 
(Control  tubes  of  B.  coli  also  failed  to  coagulate  unless  heated). 
There  was  no  further  apparent  change  until  24  to  30  days  incubation 
when  evolution  of  gas  followed  by  coagulation  and  extrusion  of  a 
clear  whey  was  noticed  in  10  of  the  cultures.  It  was  thought  that 
this  reaction  might  be  due  to  some  contaminating  organism,  but 
microscopic  examination  of  all  of  the  tubes  of  one  set  of  milk  cul- 
tures showed  only  Gram  negative  long  rods  (and  with  a  single  ex- 
ception, no  spores)  resembling  the  organism  under  consideration 

Four  cultures  were  inoculated  into  milk,  covered  with  melted 
paraffine  and  heated  at  80°C.  for  10  minutes  (the  so-called  sporo- 


336  JACK   J.    HINMAN,   JR.,    AND   MAX  LEVINE 

genes  test).  The  litmus  was  reduced,  there  was  no  coagulation  of 
the  medium  and  no  apparent  change  in  10  days  at  37°C.  •  At  this 
time  the  milk  coagulated  on  heating,  and  melting  the  paraffine  seal 
by  gently  heating  showed  that  there  was  but  a  very  small  amount  of 
gas  developed  in  the  medium.  The  organism  does  not  give  the 
characteristic  B.  sporogenes  or  B.  Welchii  test. 

INDOL  FORMATION]  ; 

Tests  for  indol  were  made  on  all  strains  in  7  days  culture  of  broth 
and  peptone  with  negative  results. 

NITRATE   REDUCTION 

All  cultures  reduced  nitrates  to  nitrites  when  grown  in  nitrate 
broth  for  5  days  at  37°C. 

FERMENTATION    OF    CARBOHYDRATES 

Glucose  neutral  red  broth.  Four  cultures  and  B.  coli  as  a  control 
were  inoculated  into  (a)  freshly  heated  neutral  red  glucose  broth 
and  (b)  old  unheated  neutral  red  glucose  broth,  in  Smith  tubes, 
and  incubated  at  body  temperature. 

In  the  freshly  heated  medium  B.  coli  formed  25  per  cent  gas  in 
24  hours,  but  there  was  no  reduction  of  the  neutral  red,  while  in  the 
closed  arm  of  the  tubes  of  the  old  unheated  medium  the  indicator 
was  reduced  to  a  canary  yellow  as  is  to  be  expected  for  B.  coli. 

The  test  cultures  reacted  in  a  similar  manner,  i.e.  the  dye  was 
reduced  in  the  unheated  medium  but  not  in  the  same  medium  which 
had  been  freshly  heated.  The  gas  formation  was  very  meagre,  in 
no  instance  being  more  than  5  per  cent.  There  was  no  change  in 
48  hours. 

Glucose  peptone  phosphate.  Seven  strains  and  B.  coli,  as  a  control, 
were  tested  for  acid  and  gas  production  in  0.5  per  cent  peptone, 
glucose,  dipotassium  phosphate  in  Smith  fermentation  tubes. 

The  results  are  indicated  in  table  2. 

Starch  peptone  water.  Five  strains  were  grown  in  1  per  cent  arrow- 
root starch  peptone  water  (Andrade  indicator).  There  was  very 
vigorous  fermentation  with  acid  and  gas  production.  Both  hydro- 
gen and  carbon  dioxide  were  formed  and  the  Voges  Proskauer  reac- 
tion was  negative. 


SPORE-FORMING   ORGANISM   FROM  IOWA 


337 


Lactose  broth.  Growth  in  lactose  broth  (with  Andrade's  indicator) 
always  observed  in  Durham  fermentation  tubes  was  not  as  vigorous 
as  in  the  glucose  phosphate  or  starch  mediums  above.  After  24  hours 
there  was  usually  a  distinct  acidity  in  the  open  arm  while  the  inner 
tube  often  showed  but  little  acidity,  (usually  in  lower  end)  and  a 
small  amount  of  gas.  After  48  hours  the  entire  medium  becomes 
distinctly  acid  and  5  to  30  per  cent  gas  may  be  obtained. 

Inulin,  sucrose  and  maltose  broths  were  all  fermented  with  acid 
and  gas  production.  In  all  cases  acid  is  first  formed  in  open  arm. 
Gas  formation  was  particularly  vigorous  with  inulin.  It  seems  to 
be  a  peculiarity  of  this  organism  that  it  attacks  the  complex  carbo- 
hydrates much  more  vigorously  than  the  hexoses. 

TABLE  2 
Growth  in  Clark  and  Lubs  medium  (48  hours,  37°) 


ORGANISM 

(ANDRADE) 

GAS  PER  CENT 

C02 

H~2  FLAME  TEST 

V.  P. 

14  Fl 

+ 

20 

+ 

+ 

— 

14  F2 

+ 

5 

+ 

+ 

— 

14  Fl 

+ 

40 

+ 

+ 

— 

14  FA 

+ 

35 

+ 

+ 

— 

14  BC 

+ 

30 

+ 

+ 

— 

12-1 

+ 

45 

+ 

+ 

— 

8 

+ 

25 

+ 

— 

— 

B.  coli 

+ 

45 

+ 

+ 

— 

FERMENTATION   OF   CARBOHYDRATES   IN    SOLID   MEDIA 

Nutrient  agar  containing  1  per  cent  of  the  test  carbohydrate 
and  Andrade's  indicator  were  employed.  Incubation  was  at  37°C. 
for  10  days.  It  was  observed  that  the  more  complex  test  materials, 
starch,  inulin,  dextrin  and  glycogen  were  particularly  vigorously 
fermented  in  2  days  (as  indicated  by  the  extent  of  disintegration  of 
the  agar)  and  that  after  4  to  10  days,  the  acidity  usually  disappeared 
and  the  medium  reverted  to  a  distinct  alkaline  reaction.  Dulcitol 
alone  was  not  decomposed. 

Carbohydrates  were  tested  with  the  results  indicated  in  table  3. 

VOGES  PROSKAUER  AND  METHYL  RED  REACTION 

Tests  for  acetyl  methyl  carbinol  and  acidity  to  methyl  red  were 
made  on  all  strains  in  Clark  and  Lubs  medium  after  incubation  at 
body  temperature  for  4  days.  The  Voges  Proskauer  reaction  was 


338 


JACK  J.    HINMAN,   JR.,    AND   MAX  LEVINE 


negative  in  all  cases.  The  reaction  to  methyl  red  was  slightly  alka- 
line or  neutral.  Growth  was  particularly  vigorous  in  this  medium 
and  was  accompanied  by  much  foaming. 


GROWTH    IN    SPECIAL    MEDIA 


Uric  add.    There  was  no  evidence  of  growth  in  Koser's  uric  acid 
medium  after  4  days  at  37°C. 


TABLE 


14  UNKNOWN  STRAINS 

CONTROLS 

(ACID 

AND 

GAS) 

B.  AER- 

OGENE8 

Acid 

Gas 

Reversion 

Para  B. 

B.  coli 

Dextrose  

+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 

+ 
+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 
4- 

+ 

+ 
+ 
+  + 
+ 
+ 
+  + 
+ 
+ 
+ 
+ 
+  + 
+  + 
+  + 
+  + 

+ 
+  + 
+  + 
+  + 
+  + 

Slow  or  negative 
Slow  or  negative 
Rapid,  4  day 
Rapid,  4  day 
Rapid,  4  day 
Rapid,  4  day 
Rapid,  4  day 
Generally  rapid 
Rapid,  4  day 
4  to  10  day 
Rapid,  4  day 
Rapid,  4  day 
Rapid,  4  day 
Rapid,  4  day 

4  to  10  day 
Rapid,  4  day 

Rapid,  4  day 
Rapid,  4  day 

+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 

+ 
+ 
+ 

+ 
+ 
+ 
+ 
+ 
+ 

4- 
+ 

+ 
+ 

+ 
+ 

+ 

+ 
•f 

+ 

+ 

+ 
+ 
+ 
+ 
+ 

+ 
+ 

-H 

Levulose.  

Galactose  

Arabinose 

Mannose  

Xylose       

Rhamnose 

Trehalose  
Melezitose    

Lactose 

Maltose  

Sucrose        

Mannitol 

Glycerol 

Dulcitol     

Snlicin                         .  . 

Dextrin  

Inulin  

Starch                  .     . 

Glycogen 

Lactose  bile.  Six  cultures  were  inoculated  heavily  into  lactose 
bile  (Difco).  No  gas  was  produced  in  4  days. 

Gentian  violet  lactose  broth.  In  lactose  broth  containing  gentian 
violet  in  a  dilution  of  1 : 100,000  there  was  no  gas  or  other  evidence 
of  growth  in  5  days  at  37°C. 

RESISTANCE    TO    HEAT 

It  has  previously  been  mentioned  that  old  cultures  taken  from 
lactose  agar  and  milk  survived  a  temperature  of  92°C.  for  20  min- 
utes although  no  spores  were  visible  on  microscopic  examination. 


SPORE-FORMING   ORGANISM   FROM   IOWA 


339 


The  following  experiment  was  performed  to  check  these  observations: 
A  loop  of  a  48  hour  lactose  (Andrade)  broth  culture  was  inoculated 
into  each  of  5  tubes  of  melted  starch  agar  which  were  kept  in  a  water 
bath  at  89°  to  91°C.  After  10,  20,  30,  45  and  60  minutes,  tubes 
were  removed  from  the  bath,  cooled  and  incubated  at  37°C.  A 
Gram  stain  of  the  broth  culture  showed  it  to  be  a  pure  culture  of 
long  Gram  negative  rods  and  no  spores  were  visible. 

Plain  and  lactose  agar  culture  suspended  in  broth  were  also  heated 
as  above.     The  results  are  given  in  table  4. 

TABLE  4 
Resistance  of  aerobic  spore  forming  lactose  fermenting  bacilli  to  heat  (89-91°C.) 


GROWTH   IN  STARCH  AGAR    (48  HOURS  37°C.)* 

TIME   OF  EXPOSURE 

Inoculated  from 

Lactose  agar 

Lactose  broth 

Plain  agar 

minutes 

10 

+ 

+ 

+ 

20 

+ 

+ 

+ 

30 

+ 

+ 

+ 

45 

-f 

+ 

+ 

60 

-{- 

-f 

-}- 

Microscopic 

Gram  negative 

Gram  negative 

Gram  negative 

examination 

long  rods. 

long  rods. 

rods.    Many 

of  inoculunn 

No  spores 

No  spores 

spores 

*  Two  cultures  employed,  14B1  and  8. 

IDENTITY    OF    ORGANISM 

Meyer  in  1919  described  a  sporing  lactose  fermenter  which  he 
isolated  from  Newport  and  Covington,  Kentucky  water.  In  many 
respects  his  organism  is  strikingly  similar  to  the  one  here  recorded 
(e.g.,  dulcitol  is  the  only  carbohydrate  not  attacked)  and  we  thought 
that  possibly  they  were  the  same,  but  rather  important  differences 
have  been  observed.  The  Meyer  strain  was  non-motile,  gave  a 
positive  Voges-Proskauer  reaction,  liquefied  gelatin  rapidly,  and 
failed  to  reduce  nitrates.  The  strain  herein  described  is  actively 
motile,  negative  for  the  Voges-Proskauer  test,  liquefies  gelatine 
slowly  and  reduces  nitrates  very  vigorously. 

Two  cultures  have  been  described  in  connection  with  studies  on 
acetone  production  which  may  be  identical  with  the  organism  re- 


340  JACK   J.    HINMAN,    JR.,    AND   MAX  LEVINE 

ported  in  this  paper.  The  B.  macerans  of  Schardinger  was  isolated 
in  1904  from  potatoes.  From  the  meagre  description  available,  it 
cannot  be  differentiated  from  the  strain  under  discussion.  In  1919, 
Northrop,  Ashe  and  Senior  described  an  acetone  producing  organism, 
also  isolated  from  potatoes,  which  they  named  B.  acetoethylicum. 
It  is  said  to  differ  from  B.  macerans  in  that  the  latter  does  not  fer- 
ment galactose  and  levulose  with  NH3  salts  as  a  source  of  Nitrogen. 
We  have  not  been  able  to  obtain  cultures  of  B.  macerans  or  B. 
acetoethylicum  for  comparison  with  our  strains.  In  the  published 
reports  the  fermentation  of  lactose  (with  gas)  by  B.  acetoethylicum 
is  not  recorded  while  B.  macerans  is  said  to  form  gas  in  milk  (presum- 
ably from  lactose) .  We  will  therefore  consider  our  strains  tentatively 
as  B.  macerans. 

SANITARY    SIGNIFICANCE 

Little  is  known  as  to  the  sanitary  significance  of  B.  macerans  and 
closely  related  forms.  Such  organisms  have  been  isolated  from 
potatoes,  retting  flax,  white  flour  and  water.  There  is  no  record 
that  they  are  present  in  the  intestinal  tract  although  a  careful  search 
may  disclose  them.  Information  as  to  the  distribution  of  these 
sporing,  lactose-fermenting  forms  capable  of  growing  aerobically 
is  now  being  gathered  and  the  pathogenicity  of  the  isolated  strains 
is  also  being  investigated.  The  organism  isolated  by  Meyer  was 
non-pathogenic. 

In  a  chlorinated  water  B.  macerans  would  be  present  long  after 
the  ordinary  water-borne  pathogens  had  been  destroyed.  The 
detection  of  B.  macerans,  in  the  absence  of  organisms  of  the  colon 
group,  in  a  treated  water  should  therefore  not  be  considered  an  indi- 
cation of  danger  from  such  intestinal  disturbances  as  typhoid  fever 
or  dysentery.  The  presence  of  these  sporing  organisms  in  water 
interferes  seriously  with  the  routine  tests  for  B.  coli  with  which  they 
may  be  confused  and  is  possibly  responsible  for  the  poor  results 
sometimes  reported  in  water  purification. 

SUMMARY 

A  Gram  negative  sporing  bacillus  capable  of  fermenting  lactose 
and  growing  aerobically  was  isolated  from  two  chlorinated  surface 
water  supplies  in  Iowa. 

The  morphological,  cultural  and  physiological  characteristics 
are  detailed. 


SPORE-FORMING   ORGANISM   FROM   IOWA  341 

The  strain  resembles  markedly  that  described  by  Meyer,  differing 
with  respect  to  rate  of  liquefaction  of  gelatin,  nitrate  reduction  and 
the  Voges-Proskauer  test. 

The  organism  should  be  of  particular  interest  to  water  works 
operators  because  of  its  extreme  resistance  to  chlorination,  and  be- 
cause of  the  ease  of  confusion  with  the  colon  group  in  routine  tests 
as  ordinarily  performed.  Its  presence  in  water  may  explain  anoma- 
lous positive  colon  tests.  Information  as  to  its  source  is  particularly 
desirable. 

The  cultures  isolated  are  strikingly  similar  to  B.  macerans  and 
B.  acetoethylicum. 

NOTE  ON  PATHOGENICITY 

The  Bacillus  acetoethylicum  of  Northrup,  Ashe  and  Senior  was 
reported  non-pathogenic  to  mice. 

One  of  the  similar  organisms  isolated  in  the  course  of  this  study 
was  tested  for  pathogenicity  on  rabbits  in  the  following  manner: 

An  agar  culture  was  scraped  to  remove  the  entire  growth  on  its 
surface  and  the  material  suspended  in  physiological  salt  solution 
and  added  to  the  drinking  water  supplied  to  a  rabbit.  This  proc- 
ess was  repeated  daily  for  a  week.  The  test  animal  did  not  show 
any  untoward  symptoms. 

Another  rabbit  was  injected  intravenously  with  one  cubic  centi- 
meter of  a  live  culture  prepared  by  removing  the  growth  of  organ- 
isms from  an  agar  slant  as  indicated  above  and  suspending  the 
material  in  10  cc.  of  sterile  salt  solution.  The  animal  developed 
no  symptoms  that  would  indicate  bacterial  infection. 

We  are  therefore  of  the  opinion  that  this  organism  is  not  a 
pathogenic  form. 

REFERENCES 

ELLMS,  JOSEPH  W.  1920  Report  of  experiments  in  the  purification  of  the 
water  supply  of  Milwaukee,  Wisconsin. 

EWING,  C.  L.  1919  Presence  of  a  spore  bearing  aerobic  gas  forming  bacillus 
in  Baltimore  city  drinking  water.  Am.  Jour.  Pub.  Health,  ix,  157- 
158. 

HALL,  I.  C.,  AND  ELLEPSON,  L.  J.  1918  The  elimination  of  spurious  pre- 
sumptive tests  for  B.  coli  in  water  by  the  use  of  gentian  violet. 
Jour.  Bact.,  iii,  329-354. 

HALL,  I.  C.,  ANDELLEFSON,  L.  J.  1919  Further  studies  on  gentian  violet 
as  a  means  of  eliminating  spurious  presumptive  tests  for  B.  coli 
in  water.  Jour.  Am.  Water  Works  Assoc.,  vi,  67-77. 


342  JACK  J.    HINMAN,    JR.,    AND   MAX   LEVINE 

MEYER,  E.  M.    1918    An  aerobic  spore-forming  bacillus  giving  gas  in  lactose 

broth,  isolated  in  routine  water  examination.    Jour.  Bact.,  iii, 

9-14. 
NORTHROP,  J.  H.,  ASHE,  L.  H.,  AND  SENIOR,  J.  K.    1919    Biochemistry  of 

B.  acetoethylicum  with  reference  to  the  formation  of  acetone. 

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Note. — This  paper  is  a  contribution  from  the  Department  of  Bacteriology 
and  Pathology  of  the  State  University  of  Iowa. 


Y.C  88560 


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